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Soil salinity is an important issue, as most crop plants are low in salt tolerance. Salt tolerance, a complex, multifactorial, and multigenic process, has been known to be a quantitative trait. The identification of the salt stress responsive genes or salt tolerance genes is essential for the breeding programs. Most recent efforts have been focused on the products of structural genes (transport proteins, ion channels, enzymes of solute synthesis) while little attention were paid to the regulatory aspects of these proteins. Since the first aquaporin gene from plants was cloned and functionally expressed in 1993, there has been a growing interest in the molecular biology of MIPs (membrane intrinsic proteins) and their bearing on the biophysics of water flow across plant membranes. In the last decades, studies on Mangroves, a special kind of wood plants, grow in high-salt and flooding conditions have been concentrated almost exclusively on their physiological and ecological characteristics. Kandelia candel, one of the dominant species of mangroves along the Chinese coast, lacks salt glands or salt hairs used for removal of excess salt in other mangroves. This makes K. candel a perfect model to study the molecular mechanism of salt tolerance in mangrove plants. Using cDNA RDA, a cDNA-specific modification of genomic representational difference analysis, a series of salt responsive genes of Kandelia candel were cloned. Among these gene fragments, a 183 bp fragment (termed as SRGKC1) encoding a tonoplast intrinsic protein (TIP) in Kandelia candel (KCTIP1) was identified. Based on the sequence of SRGKC1, two gene specific primers were designed, and the 3' and 5' end of the KCTIP1 gene were obtained using the SMART RACE cDNA Amplification Kit. RACE products were purified from low-melting agarose, and sequenced directly with GSPs as the sequencing primers. A 500-bp fragment corresponding to the 3'end of this gene was obtained using the GSP1 primer, and a 690 bp fragment corresponding to the 5' end of this gene was obtained using the GSP2 primer. Two primers that flank the putative open reading frame (ORF) were designed to obtain the cDNA containing the complete ORF by RACE PCR reaction. The full-length cDNA of KCTIP1, containing a 756 bp open reading frame (ORF), was approximately 1.1 kb; the start codon was located at the nucleotides of 99-101 and stop codon at the nucleotides of 855-857 followed by a poly (A) tail. The KCTIP1 cDNA sequence in this research was released in GenBank with accession number AF521135. Using ExPASy Proteomics tools provided by EMBL, the isoelectric point and MWt of KCTIP1 are estimated as 5.77 and 26.3 kD respectively. Transmembrane prediction analysis revealed the deduced KCTIP1 protein sequence contains six transmembrane regions at amino acid residues of 20 - 42, 57 - 79, 86 - 108, 113 - 135, 142 - 164 and 217 - 239. Two highly conserved asparagine-proline-alanine (NPA) motifs were located at 85 - 87 and 199 - 201 amino acid residues respectively. KCTIP1 is also predicted to contain the Cys residue (Cys 118) that are shown to confer Hg-sensitivity in Arabidopsis gamma-TIP and delta-TIP. Similarity analysis showed that KCTIP1 shared 77% - 79% amino acid sequence identity with the TIPs from Vitis berlandieri, Brassica oleracea and Arabidopsis thaliana. Expression analyses indicated that KCTIP1 had different expression among species of Mangroves. Expressions of KCTIP1 in Kandelia candel, Rhizophora apoculata and Ceriops tagal were suppressed by salt, and were insensitive to salt stress in unknown species of Mangroves. Previous studied showed that salt conditions might result in large and rapid changes in extracellular water potential and serious disturbance to the cytoplasm. In order to compensate for this imbalance, the relative contribution of water channels to flow across the root could thus vary. K. candel is a species that is native to intertial zone of tropical and subtropical coast and is well-adapted to salt conditions. The coordinated down-regulation of aquaporins in this plant may decrease membrane water permeability and thus increase the cellular water conserva- tion during periods of salt stress. The results reported here are consistent with the postulated roles for tonoplast water channels in regulating the hydraulic permeability of the vacuolar membranes and in adjusting the water homeostasis of the protoplasm under various physiological conditions. The identification of KCTIP1 as one of salt-responsive genes implies that intracellular osmotic equilibration is a part of salt-tolerant mechanisms in Mangroves. |
Human tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) is a member of the tumor necrosis factor (TNF) family of ligands which has been reported in 1995. The TRAIL protein induces apoptosis of certain types of target cells, such as transformed cells that include but are not limited to cancer cells and virus-infected cells but the normal cells. It is a type II transmembrane protein and the extracellular domain of TRAIL is the functional domain in induction of cell apoptosis. A gene fragment encoding for the active domain of TRAIL was modified with oligo-nucleotide directed mutagenesis according to the characters of Pichia pastoris expressing vector. Arginine at the position of 149 corresponding to the amino acid residue 531 which might be a potential Kex2 protease processing sites was substituted with Lysine to prevent the expressed protein from the digestion by the protease. After proved with DNA sequencing. the modified gene fragment coding soluble TRAIL domain was inserted into the Pichia pastoris expression vector pPIC9K in the same reading frame with alpha-factor secreting signal peptide. The recombinant plasmid pPIC9K - TRAIL was transferred into P. pastoris cell by spheroplast transformation. The recombinant yeasts were identified by antibiotic G418 and Southern dot blot. The transformants (His+ Mut(s)) containing multi-copy gene fragment of TRAIL were selected with increasing concentration of G418 and induced with 0.5% methanol in shaking flask to expression the active domain of TRAIL. After inducing for 3 - 4 days, the proteins in the culture supernatant was assayed with SDS-PAGE and Western blot. Two expressed protein bands whose appearant molecular weight were 19kD and 38kD, respectively, could be specifically recognized by polyclonal antibodies against human TRAIL. The 38kD protein might be a dimers of TRAIL in the culture supernatant. The amount of expressed foreign protein made up to 36% of the total proteins in the culture suprenatant. Biological activity assay, in vitro, indicated that the expressed protein could induce tumor cells apoptosis. |
Hepatitis C virus (HCV) is an important human pathogen that causes chronic liver disease worldwide. It is desirable to develop vaccines to prevent HCV infection, or at least to prevent progression to chronicity. We once constructed an optimized hepatitis C virus core and envelope 2 fusion antigen DNA vaccine, which could induce humoral and cellular immune responses against HCV core and E2 protein in BALB/c mice efficiently. Flt3 (Fms-like tyrosine kinase 3) -ligand has been identified as an important cytokine for the generation of professional antigen-presenting cells, particularly dendritic cells. We reasoned that a DNA vaccine coexpressing the antigen and FL may activate immune responses more effectually. In this study, The influence of FL on this HCV DNA vaccine was evaluated. The cDNA encoding signal peptide and extracellular domain of murine FL was inserted into the plasmid pST-CE2t, and the resulting plasmid pST-CE2t/FL was transfected into COS7 cells. The HCV core and E2 protein were detected by Western blotting, and the soluble murine FL was detected by ELISA. Eight-week-old female BALB/c mice were inoculated intramuscularly with 100 microg pST-CE2t, pST-CE2t/FL or mock vector, respectively, and boosted at the same dosage 3 weeks later. Anti-HCV core and E2 total IgG and isotypes were measured at weeks 1,3,5,7. Splenocyte proliferative response to recombinant HCV core and E2 protrein were detected at week 7. SP2/0 cells expressing HCV core protein were used as target cells for the detection of cytotoxic T lymphocyte (CTL) response. Western blot analysis showed that a protein band with molecular weight about 70 kD from lysate of COS7 cells transfected with plasmid pST-CE2t/FL could be detected by anti-HCV core or E2 monoclonal antibodies, which indicated that pST-CE2t could express glucosylated HCV core and E2 fusion protein. Murine FL could be detected in the culture supernatant of COS7 cells transfected with pST-CE2t/FL. Plasmid pST-CE2t immunized mice developed higher anti-HCV core and E2 IgG seroconversion rates and titers than pST-CE2t/FL group did at different various times, but the IgG2a/IgG1 ratio of anti-HCV E2 protein in pST-CE2t/FL group is much higher than pST-CE2t group. Splenocytes from pST-CE2t or pST-CE2t/FL immunized mice could proliferate with stimulation of HCV core or E2 protein in vitro, although pST-CE2t/FL group showed much stronger response. Splenocytes from mice immunized with pST-CE2t/FL induced 79.03% +/- 9.95% of target cell lysis at the effector/target ratio of 100:1, which was significantly greater than the lysis (62.2% +/- 8.62%) observed in mice immunized with pST-CE2t. Our data demonstrated that the incorporation of FL can preferentially enhance the cellular response to this HCV fusion antigen DNA vaccine. In contrast, HCV specific antibodies were inhibited by FL in vaccinated mice. More and more data supports that recovery from acute HCV infection may depend upon the generation of broad-based cellular immune responses to viral proteins. So, FL may be of potential value as an adjuvant in the development of DNA-based immunization for prophylactic and therapeutic vaccine against HCV infection. |
Hydantoin-utility-enzyme is widely used in enzymic production of various amino acids. One of its component, carbamoylase, is responsible for the conversion of N-carbamylamino acids to corresponding amino acids, which is crucial for the stereoselectivity and rate limiting. To improve the production of the enzyme, an L-N-carbamoylase gene from Arthrobacter BT801, a hydantoinase producting strain being able to convert 5-benzylhydantoin to phenylalanine, was cloned into E. coli. The gene was highly expressed in E. coli M15 under control of T5 promoter. A protein band about 44kD was detected by SDS-PAGE in the recombinant cell lysate. The objective product, which is principally in soluble form, represented 40% of total cell protein. The N-carbamoylase specific activity of the recombinant M15/pQE60- hyuC is 53 times higher than that of Arthrobacter BT801. The total biotransformation activity increased 8.1 times when. M15/pQE60-hyuC was added into the Arthrobacter BT801 reaction system. The successful expression of the enzyme is significant for the application of the hydantoinase producing strain or the enzyme thereof. |
Gamma-linolenic acid (GLA, C18:3delta6.9.12) is nutritional and important polyunsaturated fatty acid in human and animal diets. GLA play an important role in hormone regulation and fatty acid metabolization. Furthermore it is also the biological precursor of a group of molecules, including prostaglandins, leukotrienes and thromboxanes. Vast majority of oilseed crops do not produce GLA, but linoleic acid (LA, C18:2delta9.12) as its substrate. GLA is only produced by a small number of oilseed plants such as evening promrose ( Oenotheera spp.), borage (Borago officinalis) and etc. delta6-fatty acid desaturase (D6D) is the rate-limiting enzyme in the production of GLA. It can convert from linoleic acid to linolenic acid. To produce GLA in tobacco, plant expression vector was first constructed. To facilitate preparation of plant expression constructs, flanking Xba I and Bgl II restriction enzyme sites were added to the coding region of clone pTMICL6 by PCR amplification. pTMICL6 contains delta6-fatty acid desaturase gene cloned from Mortierella isabellina which is an oil-producing fugus. The PCR product was purified and subcloned into the plant expression vector pGA643 to generate the recombinant vector pGAMICL6 which contains the ORF of the D6D gene of Mortierella isabellina, together with regulatory elements consisting of the cauliflower mosaic virus 35S promoter and the nopaline synthase (nos) termination sequence. The plasmid pGAMICL6 was transformed into Agrobacterium tumefaciens strain LBA4404 by method of freeze thawing of liquid nitrogen. Transformants were selected by plating on YEB medium plates containing kanamycin and streptomycin and grown overnight at 28 degrees C, then transformants were further identified by PCR. The positive transformant containing the plant expression vector pGAMICL6 was transformed into tobacco ( Nicotiana tabacum cv. Xanthi) via Agrobacterium infection. Transgenic plants were selected on 100 microg/mL kanamycin. Plants were maintained in axionic culture under controlled conditions. Total nucleic acids were extracted and purified from anti-kanamycin transgenic tobacco and were analysed by PCR. 48 out of 80 transgenic plants were positive, in other words, transformation efficiency is 60% . This shows that Mortierella isabellina D6D gene is transformed into tobacco. Genomic DNA from PCR positive transgenic tobacco plants was digested with Hind III restriction enzyme and fractionated by agarose gel electrophoresis. Southern blotting was performed with strandard procedures for vacuum transfer of nucleic acids to nylon membrane. The probe was delta6-fatty acid desaturase gene from M. isabellina, which was labeled with DIG-dUTP via random-primed labeling. Hybridization and immumological detection were carried out the kit of DIG detection. The result shows single hybridizing bands in each of the transgenic tobacco plants DNA, but no hybridization was observed to non-transgenic tobacco. This indicates that delta6-fatty acid desaturase gene is integrated into the genome of transgenic tobacco. To provide further evidence that the introduction of the M. isabellina cDNA into the tobacco genome was responsible for the novel desaturation products, total RNA was isolated from GLA-positive transgenic tobacco plants via both PCR and Southern blotting and separated by electrophoresis through 1% formaldehyde agarose gel. Northern blotting including probe labeling, hybridization and detection was the same as Southern blotting in operation approach. A positive hybridization signal of identical mobility was obtained from RNA isolated from the transgenic tobacco plants, but not from the control tobacco plant. At last, total fatty acids extracted from the positive transgenic tobacco were analyzed by gas chromatography (GC) of methyl esters to confirm the transgenic tobacco containing a functional delta6-fatty acid desaturase gene. The result shows that two peaks were observed in the chromatogram of FAMes. GLA and octadecatetraenoic acid (OTA, C18:4delta6.9.12.15) respectively have 19.7% and 3.5% of the total fatty acids in the transgenic plant. The presence of both GLA and OTA indicates that the delta6-fatty acid desaturase used both linoleic acid and a-linolenic acid (ALA, C18:3delta6.9.12.15) as substrates, and this may be responsible for the decrease in ALA observed in the transgenic line. That was the first report about the expression of M. isabellina delta6-fatty acid desaturase gene in tobacco. All results mentioned above have laid the foundation of the thorough studying on an breeding transgenic oilseeds containing GLA to change the fatty acid composition of conventional oilseeds, it is significant to study on regulation mechanism of fatty acid desaturase. |
The NF-kappaB transcription factor plays important regulatory roles in inflammation, apoptosis, immune and stress responses. IkappaB kinase (IKK) composed of two catalytic subunits and a regulator subunit, acts as a key component of NF-kappaB activation pathway through phosphorylation of IkappaB, the inhibitor of NF-kappaB. CIKS (connection to IKK and SAPK), a newly identified cellular protein, is involved in NF-kappaB and JNK activation. Although it has been known that CIKS interacts with IKK complex, and activates both IKK and SAPK when overexpressed; the underling mechanisms are poorly understood. To better understand the physiological roles of CIKS, we have screened human HeLa MATCHMAKER cDNA library for new binding partners of CIKS by using the yeast two-hybrid system with truncated CIKS (151-574) as the bait. The yeast strain AH109 was sequentially transformed with the bait plasmid and the library. The transformants were screened on SD(-Leu/-Trp/-His/-Ade/ + X-alpha-gal)selective plates. Positive clones were restreaked on SD(-Leu/-Trp / + X-alpha-gal)plates three times to allow loss of some of the AD/library plasmids while maintaining selective pressure on both the DNA-BD and AD vectors. After 3 screenings on SD(-Leu/-Trp / + X-alpha-gal), the positive clones were further verified on SD(-Leu/-Trp/-His/-Ade/ + X-alpha-gal) plates. The inserts in AD/library plasmids were amplified by PCR and PCR products were characterized by Hae III digestion to eliminate the duplicates. After screening in selective plates, the positive AD/library plasmids were rescued via transformation of E. coli HB101 and the interactions of CIKS (151-574) with positive AD/library plasmids were further assessed by yeast two-hybrid analysis. Finally, the DNA sequences of the positive AD/library plasmids were determined and BLAST analysis against the databases was performed. The BLAST results indicate that the inserts in the positive plasmids encode RIKEN cDNA 473340F03, PLAC8, CD27BP (Siva-1), CDC5L, SnRNP smB, and DVL2. CDC5L is a key component of the multi-protein complex essential for the formation of pre-mRNA splicing product and is not required for spliceosome assembly. A role for CDC5L in the cell division cycle has been precious suggested as its overexpression of this protein in mammalian cells leads to a shortening G2 phase of the cell cycle, and a negative-dominant mutant of CDC5L lacking the N-terminal activation domain delays the G2 phase cell's entry into the mitosis. It has been reported that SnRNP smB participates in pre-mRNA splicing and CD27BP (Siva-1) binds to and inhibits BCL-XL-mediated protection against UV radiation-induced apoptosis and regulates T cell homeostasis. The functions of RIKEN cDNA 473340F03, PLAC8 and DVL2 are unknown. It has been suggested that CIKS functions as a critical component for cross-talk between NF-kappaB and JNK signaling pathways. IKK subunits, which have been demonstrated to interact with CIKS, were not shown up in this experiment. We speculate that the truncated CIKS (151-574) bait may not contain the binding domain that mediates the interaction of IKK subunits with CIKS. Taken together, the above results suggest that CIKS may play a role in cell regulation through interacting with various cellular proteins. Further investigations are required to characterize these interactions. |
(R)-chlorprenaline, a selective activator of beta2 receptor and an effective drug for bronchitis and asthma, is industrially prepared from (R)-2'-chloro-1-phenyl-ethanol. In this communication, we describe (1) the identification of Saccharomyces cerevisiae B5 as an effective host for stereoselective reduction of 2'-chloroacetophenone to (R)-2'-chloro-1-phenyl-ethanol; (2) the presence of ethanol enhances the conversion; and (3) the biochemical factors that effect the yield of the product. Among the four yeast strains capable of reduction 2'-chloroacetophenone to (R)-2'-chloro-1-phenyl-ethanol we screened, Saccharomyces cerevisiae B5 showed the highest activity and stereoselectivity, and was used for the subsequent study. The effect of the presence of methanol, ethanol, 2-propanol, 1-butanol, glucose, glycerol and lactic acid was first investigated, as it was previously reported that they increased the yield and stereoselectivity of the reaction. The addition of the co-substrate methanol, ethanol, 2-propanol, 1-butanol, glucose and glycerol favored the formation of the 2'-chloroacetophenone to (R)-2'-chloro-1-phenyl-ethanol. Lactic acid inhibited the enzyme activity. Ethanol is the best co-substrate among the seven co-substrates and under the optimum concentration of 5% , the yield of (R)-2'-chloro-1-phenyl-ethanol was increased from 17% to 74%. The oxidation of ethanol regenerates NADH required for the reduction. The effects of the reaction time, pH, cell concentration, substrate concentration and temperature on the reduction were investigated next. The enantiometric excess of (R)-2'-chloro-1-phenyl-ethanol reached 100% under the optimal condition: pH8.0, 25 degrees C and 5% ethanol. The product yield went up with the increasing Saccharomyces cerevisiae B5 concentration and reached 100% when the cell dry weight was 10.75 mg/mL and 2'-chloroacetophenone was 6.47 mmol/L. The yield of (R)-2'-chloro-1-phenyl-ethanol decreased sharply with the increase of substrate concentration, as the high concentration of substrates is toxic to the cell and inhibits the activity of reductases. The aerobic cultivation of the yeast and shaking during the reaction increased the yield of (R)-2'-chloro-1-phenyl-ethanol. The yeast can be reused up to 15 times. This research paves the way for economical preparation of chiral 2'-chloroacetophenone to R-2'-chloro-1-phenylethanol. |
In order to obtain an adequate supply of alpha-galactosidase for research and practical use, the fermentation, purification and identification of the recombinant coffee bean a-galactosidase were carried out. Baffled flasks containing 100mL BMGY were inoculated with the pPIC9K-Gal/GS115 strain and allowed to grow at 30 degrees C, 250- 300r/min until a maximum optical density at 600nm (OD600) between 2.0 to 6.0 was attained. Entire 400 mL seed culture was transferred aseptically to the 5-liter fermenter, which contained 4 liter sterilized basal salts medium and 4% glycerol. The batch culture grew at 30 degrees C, pH 5.0 until the glycerol was completely consumed, and a glycerol feed was initiated to increase the cell biomass prior to induction with methanol. The culture was centrifuged at 8000 x g and the supernatant was collected. Following ultrafiltration, the retentate was balanced in 20 mmol/L sodium formicate buffer, pH 3.8 and loaded onto a cation-exchange column, HiTrap SP. The column was washed with the same buffer and bound proteins were eluted with 1 mol/L NaCl. The fractions containing recombinant a-galactosidase were pooled and concentrated with PEG20 000. Subsequently, the biochemical properties of the enzyme were determined with typical methods. At last, the fresh human blood A and B erythrocytes were incubated with the purified alpha-galactosidase at 26 degrees C for 2 4 hours. Hemagglutinins were assayed by the standard method. After an elapsed fermentation times (EFT) of 18h, the fed-batch phase was initiated to increase the cell biomass. A cellular yield of nearly 200 g/liter wet cells was achieved when induction was initiated. 72h later, the alpha-galactosidase activity against artificial substrate PNPG (PNP-alpha-galactopyranoside) achieved 36 000u per liter culture. The crude fementation supernatant contained few impurities as detected by SDS-PAGE. The supernatant was purified by cation-exchange chromatography, the target alpha-galactosidase was eluted with 40% 1mol/L NaCl and showed a 41kD band on SDS-PAGE. After concentration, the final recovery was about 41%. The Michaelis constant of the recombinant alpha-galactosidase was determined as 0.275 mmol/L, which slightly lower than the nature enzyme and suggested a higher affinity with specific substrate. When human blood type B erythrocytes pretreated with 100u/mL recombinant alpha-galactosidase reacted with bood type B antiserum, no hemagglutination occurred. This suggested that the B antigens had been removed by the enzyme successfully. These results demonstrated that the recombinant alpha-galactosidase could be produced in largescale and made it possible to explore the application of alpha-galactosidase in more fields. |
Fruit ripening is associated with a number of physiological and biochemical changes. They include degradation of chlorophyll, synthesis of flavor compounds, carotenoid biosynthesis, conversion of starch to sugars, cell wall solublisation and fruit softening. These changes are brought about by the expression of specific genes. People are interested in the molecular mechanism involved in the regulation of gene transcription during fruit ripening. Many fruit-specific promoters such as PG, E4, E8, and 2A11 have been characterized and shown to direct ripening-specific expression of reporter genes. AGPase plays the key role in catalyzing the biosynthesis of starch in plants. It is a heterotetrameric enzyme with two small subunits and two large subunits, which are encoded by different genes. In higher plants, small subunits are highly conserved among plant species and expressed in all tissues. And the large subunits are present at multiple isoforms and expressed in a tissue-specific pattern. In fruits, the expression pattern of the large subunits varies with plant species. That made it important to study the transcriptional regulation of the large subunits of AGPase in different plant species. Northern-blot analysis indicates in watermelon, an isoform of the large subunits Wml1 expressed specifically in fruits, not in leaves. The 5' flanking region of Wml1, which covers 1573bp, has been isolated through the method of uneven PCR. And transient expression assay has shown that the 1573bp (named WSP) can direct fruit-specific expression of GUS gene. Our goal in this study was to scan the promoter region for main regulatory regions involved in fruit-specific expression. A chimaeric gene was constructed containing the WSP promoter, the beta-glucuronidase (GUS) structural sequence as a reporter gene and the nopaline synthase polyadenylation site (NOS-ter). The plasmid pSPA was digested with Hind III + Hinc II and promoter fragment of 1573bp (from 180bp to 1752bp) was cut out and cloned into Sma I sites of pBluescript SK(-), to produce pBSPA-16. The same insert was then cut out with Hind III + BamH I, and ligated with transient expression vector pBI426 digested by HindIII + Bgl II to produce pISPA-16. Three 5'-end deletions of the promoter were obtained and fused to GUS gene in plant transient expression vector pBI426: the 1201bp fragment (from 551bp to 1752bp) was generated by digestion of pBSPA-16 with BamH I + SnaB I, the 898bp fragment (from 854bp to 1752bp) by BamH I + EcoRV. Both fragments were ligated with pBluescript SK(-) digested by BamH I + Sma I, to produce pBSPA-12 and pBS-PA-9. The inserts were cut out with HindmIII + BamH I and ligated with pBI426 digested by Hind III + Bgl II, to produce pISPA-12 and pISPA-9. The 795bp fragment (from 957bp to 1752bp) was generated by digestion of pSPA with Hinc II + EcoR I, promoter fragment was cut out and cloned into Sma I sites of pBluescript SK(-), to produce pBSPA-8. The same insert were cut out with Hind III + BamH I, and ligated with transient expression vector pBI426 digested by Hind III + Bgl II. The 1573bp fragment and three 5'-end deletions were delivered into watermelon leaf, stem, flower and fruit of different development stages (5, 10, 20 days after pollination) via particle bombardment using a biolistic PDS-1000/He particle gun. Bombardment parameters were as follows: a helium pressure of 1200 psi, vacuum of 91432.23Pa, 7 cm between the stopping screen and the plate. Histochemical assay were done on all the tissues bombarded after incubation for 2 days. The 1573bp fragment had the strongest promoter activity, and can induce GUS expression in fruits of 5 and 20 days after anthesis and flowers, but not in fruits of 10 days after anthesis, leaves and stems. Fragments of 1201bp and 898bp can induce GUS expression only in fruits of 20 days after anthesis, and with lower expression levels than 1573bp. Fragment of 795bp was not able to direct GUS expression in any of the tissues bombarded (data not shown). It can be concluded that of the 1573bp, 1201 bp, 898bp Wml1 5'flanking regions include the necessary information directing fruit-specific expression. Deletion from 180bp to 551bp doesn't affect the fruit-specificity of the promoter, but lowered the expression level. There may be some cis-acting elements located in this region, which can enhance external gene expression in later stages of fruit development. Deletion from 854bp and 958bp led to loss of GUS expression. This region includes the necessary information needed for gene expression as well as the regulatory elements for fruit-specific transcription. |
Cellulosic material is the most abundant renewable carbon source in the world. Cellulose may be hydrolyzed using cellulase to produce glucose, which can be used for production of ethanol, organic acids, and other chemicals. Cellulase is a complex enzyme containing endoglucanase (EC 3.2.1.4), exoglucanase (EC 3.2.1.91) and cellobiase (EC 3.2.1.21). The hydrolysis of natural cellulose to glucose depends on the synergism of these three components. The mostly used cellulase produced by Trichoderma reesei has high activity of endoglucanase and exoglucanase, but the activity of cellobiase is relatively low. Therefore, improving the activity of cellobiase in cellulase reaction system is the key to enhance the sacchrification yield of cellulosic resources. Aspergillus niger LORRE 012 was a high productivity strain for cellobiase production. It was found that the spores of this strain were rich in cellobiase. In this work, the cellobiase was immobilized efficiently by simply entrapping the spores into calcium alginate gels instead of immobilizing the pure cellobiase proteins. The immobilized cellobiase was quite stable, and its half-life was 38 days under pH 4.8, 50 degrees C. The thermal stability of the immobilized cellobiase was improved, and it was stable below 70 degrees C. The suitable pH range of the immobilized cellobiase was pH 3.0 - 5.0, with the optimal pH value 4.8. The Km and Vmax value of the immobilized cellobiase were 6.01 mmol/L and 7.06 mmol/min x L, respectively. In repeated batch hydrolysis processes, 50 mL of substrate (10 g/L cellobiose) and 10 mL of immobilized beads containing cellobiase were added into a 150 ml flask. After reacting at pH 4.8, 50 degrees C for several hours, the hydrolysate was harvested for assay, and the immobilized beads were used for the next batch hydrolysis with the fresh substrate. This process was repeated, and the yield of enzymatic hydrolysis kept higher than 97% during 10 batches. The continuous hydrolysis process was carried out in a column reactor (inside diameter 2.8 cm, inside height 40 cm) packed with the immobilized beads. Using 10 g/L cellobiose as substrate, the hydrolysis yield reached 98% under 0.4 h (-1) dilution rate and pH 4.8, 50 degrees C. After corncob was treated by 1% dilute acid, the cellulosic residue (100 g/L) was used as substrate, and hydrolyzed by the cellulase (15 IFPU/g substrate) from Trichoderma reesei, at pH 4.8, 50 degrees C for 48 h. The concentration of reducing sugar in the hydrolysate was only 48.50 g/L (hydrolysis yield 69.5%). When the hydrolysate was further treated by the immobilized cellobiase, the cellobiose was hydrolyzed into glucose, and the feedback inhibition caused by the cellobiose accumulation disappeared sharply. By the synergism of immobilized cellobiase and the cellulase from T. reesei left in the hydrolysate, other oligosaccharides were mostly converted to monosaccharides. At 48 h, the reducing sugar concentration was increased to 58.78 g/L, the hydrolysis yield of the corncob residue was improved to 84.2%, and the ratio of the glucose in the total reducing sugar was increased from 53.6% to 89.5%. The reducing sugars converted from corncob could be used further in the fermentation of valuable industrial products. This research results were meaningful in the conversion and utilization of renewable biomass. |
S1 nuclease (from Aspergillus oryzae) is a specific enzyme to degrade single stranded DNA or RNA molecules. It has been reported to be able to convert superhelical circular DNA molecules into open circle or linear forms under certain conditions, but this function has not been well explored. In order to use the action of S1 nuclease to linearize circular DNA and develop a novel way of cloning microcircular DNAs, the pUC19 was used to investigate the relationship between the linearization efficiency of S1 nuclease and the amount of enzyme used. By this way the optimal conditions for linearization of circular DNAs by S1 nuclease would be determined. 0.3u to 17u S1 nuclease per 100ng pUC19 DNA was added into a 25 microL system, respectively, to perform the reaction. The effectiveness of enzyme digestion was realized by electrophoresis in a 1.2% agarose gel. The results showed that along with the increase in enzyme amount from 0.3u to 17u a gradual decrease in the superhelical form, a gradual increase in the linear form and then in the circular form was obvious. The conversion from superhelical form to linear and circular form was directly related to the enzyme amount used. A higher proportion of linear DNA molecules was achieved by using 5 to 17u S1 nuclease per 100ng DNA. Besides, electrophoretic mobility of the S1 nuclease-linearized pUC19 was the same as that of the linear form produced by restriction enzyme digestion. According to the result of phiX174 digested by S1 nuclease it has been proposed that the enzyme cleaves first randomly on one site of one strand, thus converting the superhelical molecules into open circle form, and then on the same site of the complementary strand to produce the linear form. Therefore, the S1 nuclease-linearized DNA molecules are intact in the sense of their length and can be used for cloning. The plasmid-like DNA pC3 from cucumber mitochondria is a double stranded circular DNA molecule with about 550bp and the smallest known plasmid-like DNA in eukaryotic mitochondria. Many attempts have been made to linearize the molecule by using restriction enzymes but failed. Therefore, S1 nuclease was used to linearize pC3 based on the results obtained with pUC19. The linearized pC3 DNA molecules formed a very sharp band in a 2.5% agarose gel after electrophoresis. They were then recovered from the gel, added an "A" tail and ligated with T-vector. After transformation into E. coli JM109 cells, the positive clones were, screened by the blue-white selection. The insert was then cut using restriction enzymes EcoRI and Pst I. The result of electrophoresis shows that the electrophoretic mobility of the insert is just the same as that predicted. A 32 P-labled probe was synthesized using pC3 as the template and Southern blot analysis was carried out. The result shows that the inserted DNA is hybridized to the probe, which indicates that the cloned DNA fragment is from pC3. The sequence information of the insert shows that the plasmid-like DNA pC3 was 537bp in length. The nucleotide sequence was deposited in the GenBank (the accession number is AF522195). |
In eukaryotes protein phosphorytion is a key event. By reversible protein phosphorylation eukaryotes control many cellular processes including signal transduction, gene expression, the cell cycle etc. Phosphoproteomics involves identification of phosphoproteins and phosphopeptides, localization of the exact residues that are phosphorylated and quantitation of phosphorylation. Because protein phosphorylation is a dynamic process, and it is present at low abundance within cells, and the phosphorylated sites on proteins might vary, and mass spectrometry (MS) signals from phosphopeptides are usually suppressed etc., so phosphoprotein analysis have more difficulties than nonphosphoprotein. In this article, we outline several analysis techniques for separation, identification and quantitation of phosphorylated proteins and peptides, and discuss the progress in these techniques. At present, MS is still an essential core identification technology for phosphoproteomic studies, To search better enrichment strategies are the main challenges in this rapidly evolving field. A major goal of quantitative proteomics is precise quantification and identification of proteins in complex mixtures. A common method for quantitative proteome analysis is the stable isotope labeling method. Today there is no single method that supersedes all others techniques for Phosphoproteomic studies. With continued development of sample preparation techniques and instrumentation, it should be possible to perform a global analysis of protein phosphorylation. |
Protein splicing is a newly discovered posttranslational editing process that removes an internal protein fragment from the protein precursor. During the splicing process the internal protein fragment, intein, triggered the self-excision from the precursor protein and the concomitant ligation of the flanking protein fragments, exteins. The self-catalysis requires neither auxiliary enzymes nor cofactors and only involves four intramolecular reactions. A number of key catalytic residues in inteins and flanking fragments have been identified, which led to the development of the protein splicing process as a protein engineering tool. Controllable cleavage of the peptide bond at either the N or the C terminus of an intein has allowed the design of novel strategies for manipulation of protein and peptides. Affinity purification of recombinant proteins can be facilitated by fusion the target protein with an intein. The fusion also creates C-terminal thioester, which expands the scope of chemical ligation in protein. Inteins can be engineered in a "split and inverted" configuration to form a cyclic polypeptide consisting of the sequence linking two intein subdomains. This article summarizes the recent advance in the mechanism of protein splicing and its applications in protein purification, protein ligation and protein cyclization. |
In recent years, Bacterial resistance is more and more serious for the irrational use of antibiotics produces resistant strains and other reasons. Human are trying to solve the problem from different ways, including the study of antimicrobial peptides. Defensin is one of the most important of antimicrobial peptides. A novel antimicrobial peptide, human beta-defensin 3, was isolated and demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes. The total RNA was extracted from human tonsil and the hbetaD-3 specific cDNA sequence was amplified with RT-PCR. After sequenced, the target DNA fragment was cloned into pQE-80L vector together with the DNA fragment encoding carrier protein DHFR. The recombinant vectors were transformed into E. coli M15 and the expression was induced based on the optimal values of the IPTG concentration incubation temperature and induction time determined in the previous section. The expressed proteins were analyzed by SDS-PAGE and Western-blotting. The mass of the recombinant protein was about 40% of total bacteria protein. Isolate and purify the target protein. The recombinant hbetaD-3 fusion proteins possess the antimicrobial activity to staphylococcus aureus, multiresistant staphylococcus aureus (only vancomycin-sensitive) and Candida albicans in the assay of drug susceptibility. Advanced study can be continued based on our experiments. |
The aim of this study is to develop monoclonal antibody against human hepatocyte growth factor activator inhibitor 1 (HAI-1) for future study of HAI-1. The cDNA fragments of human hepatocyte growth factor activator inhibitor 1 (HAI-1) were subcloned to construct GST-HAI-1 fusion protein expression vectors. The vectors were transformed into E. coli and fusion protein expression was induced by IPTG. The GST-HAI-1 fusion proteins were separated on preparative SDS-PAGE and recovered by electroelution, and used to immunize BALB/c mice. Hybridomas producing monoclonal antibodies against human HAI-1 were prepared by cell fusion technique and characterized by ELISA, Western Blot and immunohistochemical staining. One hybridoma cell line, ZMC6, was obtained, which produces specific antibody against the expressed GST-HAI-1 fusion protein. The monoclonal antibody recognizes both the membrane-type and secretory-type HAI-1 proteins of colorectal tissue. The successful development of anti-HAI-1 antibody provides a powerful tool for further investigation on HAI-1's function. |
Translocating protein and translocating peptides have therapeutic potential against tumors by exposing the cytotoxic domains of toxic proteins to the cell cytosol. The aim of this study is to investigate the effect of N-terminally fused PE translocating peptides on granzyme B (GrBa) activity. PE II-GrBa fusion protein genes were constructed by replacing N-terminal signal and acidic dipeptide sequence of human granzyme B gene with two truncated translocating sequences of Pseudomonas exotoxin A (PE II aa 280-364/358) by recombinant PCR, and then cloned into pIND inducible expression vector. The resulting pIND-PE II-GrBa expression vectors were co-transfected with assistant plasmid pVgRXR into HeLa cells through lipofectamine, followed by selection on G418 and zeocin. The resistant cells were collected and induced with ponasterone A. Western blot analysis demonstrated that ponasterone A induction caused the expression of PE II-GrBa fusion proteins, and indirect immunofluorescence detected giant sized multinucleated cells, suggesting cytoskeletal and mitotic abnormalities as reported in our previous studies. Western blot, enzymatic activity assay and cell counting analysis indicated that two types of PE II-GrBa fusion proteins were capable of cleaving both endogenous and exogenous substrates of granzyme B, and inhibiting the growth of cells. The PE II (aa 280-358)-GrBa was shown to have higher serine protease activity and stronger growth inhibitory effect. Such inhibition was presumably associated with G2 arrest as determined by cell cycle analysis. These data prove that PE II-GrBa fusion proteins have cell inhibitory effect similar to GrBa, and that the shorter PE-derived peptide exerts less influence on GrBa activity. This study helps to optimize the construction of recombinant protein comprising translocating peptides and cytotoxic molecules for tumor cell killing. |
Using overlapping and mutant oligonucleotides as probes, gel mobility assays and competition experiments identified a sequence from -47 to -32 bp upstream of the LIM2 CAP site, which a lens protein complex bound with high affinity which appeared to bind only to the "sense" strand of the double-stranded DNA molecule. This sequence consisted of a string of four guanine residues followed by seven other nucleotides (AACCTAA) and followed by another four guanines, i.e. GGGGAACCTAAGGGG, called the Hsu element. Promoter-CAT constructs containing this sequence or mutations of the sequence indicated that the Hsu element is located within the basal promoter, and is essential for expression of the LIM2 gene. The trans factors binding to the Hsu element are present throughout development, and appear to be lens-specific. Since the LIM2 gene promoter does not contain a classic TATA box, the Hsu element may serve as the site for binding the RNA polymerase complex. |
Rhesus monkeys (5 in each group) were inoculated with recombinant fusion protein of cholera toxin B subunit and multi-valent epitopes of Plasmodium falciparum intranasal or intramuscular (i.m.). Immune-responses and protective effect were evaluated. The antibody titer (Geometry mean) against CTB reached 1:512 (intranasal) and 1:10000 (i.m.) 14 day after 3rd immunization, and antibodies against P. falciparum were also elucidated, the titers in i.m. group were also significantly higher than that in intranasal group. The monkeys were challenged with 1.25 x 10(8) sporozoites of P. cynomolgi, Patent infection was observed in all 5 monkeys in control group inoculated with PBS in 10 - 14 days after challenge. Patent infection was also observed in 5 animals inoculated via intranasal and 2 animals in intramuscular group 19th days after challenge, But the infection last only 4 days in 3 animals in intranasal group and 2 animals in intramuscular group. The results demonstrated that the vaccine candidate could induce protective immune-responses in rhesus monkey against the challenge of P. cynomolgi. |
With the application of gE gene deleted pseudarabies virus (PRV) vaccine worldwide, a corresponding differential diagnosis based on gE glycoprotein is needed in the project of PRV eradication. In this study, PRV gE gene without signal and transmembrane region was amplified by PCR and cloned into pGEX-6P-1, generated pGEX-gE. After transformation of BL21 with pGEX-gE, an expressed fusion protein(about 63kD) was identified by SDS-PAGE. The recombinant proteins are produced as inclusion bodies. By changing the inductive conditions, the formation of inclusion bodies was inhibited and tended to increase the percentage of soluble recombinant protein. The antigenic reactivity of the recombinant protein was confirmed by Western blotting with polyclonal antibodies against PRV. Using purified recombinant tgE as antigen, an ELISA was developed to detect sera of PRV infected pigs and sera of pigs immunized with gE-deleted PRV vaccine. The total of 400 serum samples collected from field were comparatively tested with the tgE-ELISA and a commercial competitive ELISA based on monoclonal antibody against gE, the results indicated that the coincidental rate between the two tests is about 94%. |
The expression of cDNA encoding Tachyleus auti-lipoposaccharide (LPS) factor, which is of interest for use as a potential inhibitor of the common core subunit of Gram-negative bacterial endotoxin. First, the TALF gene was inserted into expression vectors pGEX-4T-2, pET22b and pET28a to construct recombinant expression plasmids. The recombinant plasmids were transformed to E. coli BL21 (DE3) and the expression of TALF was examined. Results show that TALF in pET22b and pET28a vectors can't be expressed. Only the fusion protein GST-TALF was expressed in E. coli BL21 existing as inclusion bodies. From 1 liter of culture, about 4mg of fusion protein GST-TALF with 91% purity was finally obtained. No apparent bactericidal activity and LPS neutralizing activity of the fusion protein GST-TALF were found. After digested with thrombin, the fusion protein GST-TALF exhibited strong bactericidal activity and LPS neutralizing activity. |
To prepare carboxyl terminus truncated human papillomavirus type 58L1 protein, and study on its in vitro bioactivity. PCR was used to amplify carboxyl terminus truncated HPV 58L1 gene, the product was inserted into the PUCMT cloning vector, preparing recombinant PFastBacHTb containing carboxyl terminus truncated HPV58L1 gene. Further more, the recombinant plasmid PfastbacHTb was used to transform DH10Bac cells, constructing recombinant Baculovirus, then the recombinant virus was successfully used to infect Sf-9 insect cells. After incubating at 27 degrees C for 72 hours, the infected cells were collected and total cellular proteins were extracted. The target protein with MW 58KD was revealed by SDS-PAGE and confirmed by Western blot. The interested protein was purified by ProBond purification system. The purified interested protein was identified to self-assemble into VLPs by Transmission electron microscope, and induce murine erythrocyte hemagglutination, indicating that the given proteins had the conformation of VLPs, collecting, HPV58L1 proteins with carboxyl terminus truncation could be efficiently expressed in baculovirus Sf-9 cells expression system, it has identical in vitro bioactivity to the wild type HPV58L1, The present study is fundmental for preparing HPV58L1 prophylactic vaccine. |
Two distinct routes (classical mevalonate pathway and a novel mevalonate-independent pathway) are utilized by plants for the biosynthesis of isopentenyl diphosphate, the universal precursor of isoprenoids (Fig. 1). Present researches indicated that taxol was synthesized mainly via non-mevalonate pathway, but not genetic evidence was showed. The second step in non-mevalonate pathway involves an intramolecular rearrangement and subsequent reduction of deoxyxylulose phosphate to yield 2-C-methyl-D-erythritol-4-phosphate, and 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) with responsibility for this reaction was considered as a key enzyme. As a tool for the isolation of genes in terpenoid biosynthesis in plants, total RNA was prepared from Taxus chinensis suspension cells, a cell type highly specialized for diterpene (taxol). A reverse transcription-PCR strategy based on the design of degenerated oligonucleotides was developed for isolating the gene encoding a gymnosperm homolog of this enzyme from Taxus chinensis. Through sequence analysis by Blast P online, the resulting cDNA showed highly homologous to 1-deoxy-D-xylulose 5-phosphate reductoisomerases, with 95% identification compared with Arabidopsis thaliana (Q9XFS9), 94% with Mentha x piperita (Q9XESO), 80% with Synechococcus elongatus (Q8DK30), 78% with Synechocystis sp. PCC 6803 (Q55663) and Nostoc sp. PCC 7120 (Q8YP49), and 73% with Synechococcus leopoliensis (Q9RKT1). Deduced amino acid sequences were also analyzed by PROSITE, ClustalX (1.81) and Phylio (3.6 alpha), and data present evidence for the existence of this deoxyxyluose phosphate reductoisomerase in Taxus chinensis. This is the first report of the dxr gene cloned from gymnosperm. |
The xylanolytic enzymes found in Thermotoga maritima showed extremely high thermostability and considerable potential in industrial application. Yet expression level of the genes encoding these enzymes was very low. The alpha-glucuronidase gene aguA from T. maritima ATCC 43589 was cloned and expressed in several E. coli strains with different vector. The alpha-glucuronidase was overexpressed in E. coli BL21-CodonPlus(DE3)-RIL with plasmid pET-28a(+), and made up about 20% of the total proteins present in the intracellular soluble fraction. The results proved the assumption that rare codons for arginine (AGA/AGG) and isoleucine (AUA) affect the expression of aguA gene from hyperthermophilic bacterium T. maritima in E. coli. Purification procedure included two steps, heat treatment and immobilized metal affinity chromatography, and over 13.5mg of pure enzyme was obrained from 1L of induced culture. The purified enzyme showed a single band on SDS polyacrylamide gel electrophoresis with a purification of 5.1 fold, and a yield of 55.1%. The optimum activity of recombinant alpha-glucuronidase was found at pH 6.0 and 85 degrees C, the enzyme retained 70% of its activity after 1 h of incubation at 85 degrees C. The induction conditions for expression of recombinant strain BL21-CodonPlus(DE3)-RIL/pET-28a-aguA were studied on induction time and duration by IPTG. The results showed that the activity of thermostable alpha-glucuronidase reach the maximum in 5-hour after inducted at the exponential phase (OD600 of 0.7 - 0.8). |
The glycine amino peptidase of Actinomucor elegans was studied in this work. For the enzyme production Actinomucor elegans was cultured with an enzyme producing medium. Then the cells were collected and subjected to enzyme purification. The glycine aminopeptidase was purified 592 times by a DEAE-Toyopearl column, a Toyopearl HW 65-C column and a Superdex 200 column subsequently and the purified enzyme had a specific activity of 14.2 u/mg. The enzyme was estimated to have molecular mass of 320kD by gel filtration and a subunit size of 56.5kD by SDS-PAGE. It hydrolyzes glycine residue containing substrates such as glycine-betanaphthylamine more efficiently than those containing other amino acid residue. Addition to Gly-betaNA, the enzyme could also hydrolyze Ala-betaNA, Met-betaNA, Leu-betaNA, Arg-betaNA and Ser-betaNA but it had no activity on the substrates such as Trp-betaNA, Pyr-betaNA, Pro-betaNA, Asp-betaNA, Lys-betaNA, Val-betaNA. It was also observed when the glycine-betanaphthylamine concentration was higher than 2mmol/L the enzyme showed a substrate inhibition, and at the 20 mmol/L the enzyme only showed about 55% activity as it showed at the 2mmol/L. Whereas no such phenomenon was observed on the other substrate such as alanine-betanaphthylamine. The optimal temperature and pH for the reaction of this enzyme is 30 degrees C and pH 8.0, respectively. The Km and Kcat of the enzyme for glycine-betanaphthylamine is 0.24 mmol/L and 100.8 s(-1), respectively. Zn2+, Cu2+ and Cd2+ suppress almost all activities of the enzyme at the concentration of 1.0 mmol/L. Based on the study of chelating reagents, GAP belongs to the metalloenzyme. When a gelatin solution was hydrolyzed with 0.5% of alkaline proteinase together with glycine aminopeptidase at 50 degrees C for 18 hours, the glycine aminopeptidase could improve the hydrolysis degree of the protease. The total free amino acid was improved about 13% and although the enzyme mainly had the activity to hydrolyze the glycine residue, individual amino acids analysis with an amino acid analyzer showed that the contents of glycine, proline, alanine, arginine and glutamate were considerably increased. The results of this study showed that the glycine aminopeptidase would be useful in the food industry. |
Chondroitinase has been used as an important tool in the study of the structure, function and distribution of glycosaminoglycans for many years. Recently, the enzyme has been reported to be a potential enzyme for chemonucleolysis, an established treatment for intervertebral disc protrasion. In this paper, a chondroitinase had been purified from the culture supernatant of Aeromonas sobria YH311 using a simple purification procedure of ammonium sulfate precipitation, QAE-Sephadex A50 ion exchange chromatography and Sephadex G-150 gel filtration. The immobilization of purified chondroitinase using sodium alginate or cellulose as carriers has also been studied. The chondroitinase obtained from Aeromonas sobria YH311 was purified 55-fold to 95.3% pure, the specific activity of the purified enzyme was 31.86u/mg and the yield was 37%. The molecular weight of chondroitinase from Aeromonas sobria YH311 was determined by SDS-PAGE to be 80kD, which was almost the same as those chondroitinase AC from Arthrobacter aurescens, Aeromonas liquefaciens and Flavobacterium heparinum. But its isoelectric point was 4.3 - 4.6, which was far lower than the microbial chondroitinase AC. After the immobilization on sodium alginate or cellulose, the properties of chondroitinase changed greatly. The optimum temperature and pH of the free enzyme were 50 degrees C and 7.0 respectively, and about 10% activity remained after heat treatment at 80 degrees C for 20 minutes, and 47% activity remained after two weeks storage at 4 degrees C. The chondroitinase immobilized on sodium alginate had the optimum temperature and pH of 40 degrees C and 7.0 respectively, about 50% activity remained after 80 degrees C heat treatment for 120 minutes and 50% remained after 30 days storage at 4 degrees C. The chondroitinase immobilized on cellulose had the optimum temperature and pH of 70 degrees C and 6.0 respectively, and more than 70% activity remained after heat treatment at 80 degrees C and 30 days storage at 4 degrees C. The yield of the immobilization was very low, with 18.56% for alginate and 18.86% for cellulose. |
In silkworm moth the colleterial gland markedly enlarged due to the secretion and accumulation of glue like substances before adult emergence. However, the Ng mutant female moth only secreted little glue-like substance and laid loose eggs naturally. In the present experiment, it was extracted the proteins of secretory part of the variety E981 and its Ng mutant line and analyzed by two-dimensional electrophoresis. More than 700 protein spots were resolved both in two samples and most of the proteins were distributed in the area from 30 kD to 70 kD and pH 4 - 8. Through the comparison and analysis, it was found that 4 proteins were only expressed in E981 and 2 proteins were only expressed in Ng mutant. Furthermore, there are about 29 proteins were expressed higher in 981 and about 15 proteins expressed volume were higher in Ng mutant. These differential proteins may be have some relations with the Ng mutant form and directly lead to the Ng mutant can't secret the glue-like substance. |
Protein disulfide isomerase-related protein A (PRPA) was highly expressed (about 34%) in Escherichia coli by inserting the whole PRPA cDNA into the vector pET23b. After expression, the purified protein was acquired through ammonium fractional precipitation and Bio-Rex 70 chromatography. PRPA shows low disulfide isomerase activity (only about 1/250 of that of hPDI), decreases the reactivation yield of denatured and reduced lysozyme either in redox and non-redox Hepes buffer or redox PBS buffer and facilitates the aggregation of denatured and reduced lysozyme. Fluorescence spectra of PRPA indicate that PRPA has more hydrophobic groups at surface than that of hPDI, and which can be used to explain why PRPA has anti-chaperone activity during the refolding of denatured and reduced lysozyme. |
Heat shock protein gp96 is a glycoprotein which was found several years ago. Besides its function as a molecular chaperone, it is also reported to play important roles both in innate immunity and adaptive immunity. Gp96 can stimulate the maturation of antigen presenting cells (especially dendritic cells) and the secretion of cytokines. Gp96 and its associated peptides could stimulate peptide specific cytotoxic T lymphocyte reaction (CTL), which was very promising in the designing of anti-virus and anti-tumor vaccines. However the expression level of whole length gp96 was relatively low in E. coli and the purity of gp96 are not very suitable for further study. We successfully cloned the carboxy terminal fragment (560aa-751aa) of murine gp96 into the pGEX-6p-1 vector and expressed in BL21 strain. This fragment contains the peptide binding domain and the dimerization domain. After purification, the recombinant fusion protein was cleaved with the PreScission Protease and analyzed by Gelfiltration. The results show that this fragment may be related to the dimerization of gp96 and make an foundation for further investigations of the protein. |
Scansite is a short linear motif-based scanning approach established in the latest two years. It's accessible over the World Wide Web and can be used to identify sequence motifs likely to be phosphorylated by specific protein kinases or likely to bind to specific protein domains such as 14-3-3, SH2 and SH3 domains. The usage and function of the potent approach were reviewed and compared with previously established tools for phosphorylation prediction. The facing problems and application outlook of Scansite in prediction of cell signaling networks within proteomes were also presented. |
Aptamers are oligonucleotides derived from an in vitro evolution process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Aptamers specially binding to targets could recognize and inhibit the function of targets. Using this method, many powerful antagonists of cytokines have been found. In order for these antagonists to work in animal models of disease and in humans, it is necessary to modify the aptamers. First of all, 2'-F, 2'-NH2 and 2'-CH3O modifications of nucleoside triphosphates could prolong half-lives in blood. Aptamers can be kept in the circulation from hours to days by conjugating them to higher molecular weight vehicles. After modified, conjugated aptamers are injected into animals, they inhibit physiological functions known to be associated with their target cytokines. Exhibiting binding characteristics comparable to or even better than monoclonal antibodies, these ligands can be used as detection probes, highly efficient inhibitors of protein function or specific competitors in high-throughput screening (HTS) assays. Recently several aptamers of cytokines have been characterized. Some of them have been used as diagnostic agent for the detection of target cytokines. The first aptamer that has proceeded to phase II clinical studies is NX-1838, an injectable angiogenesis inhibitor that can be potentially used to treat macular degeneration-induced blindness. Aptamers will be versatile tools that can greatly enhance the efficiency of modern diagnose and therapy development. |
To investigate the function of ALK3 gene, the gene regulation and the signaling pathway related to ventricular septum defect during heart development. The model mice with ALK3 gene knock-out via alpha-MHC-Cre/lox P system were bred. The mRNA expression level of control group was compared with that of experiment group and ALK3 downstream genes were screened using PCR-select cDNA subtraction microarray. The mRNA of control group was extracted from E11.5 normal mouse hearts, and that of experiment group, from E11.5 hearts of mice with alpha-MHC Cre(+/-) ALK3(F/+) genotype. It was found that the mice with ALK3 gene knock-out produced heart defects involving the interventricular septum. The platelet-activating factors acetylhydrolase and the transcription factor Pax-8 and so on, were down-regulated. However, the Protein Tyrosine Kinase (PTK) of Focal Adhesion Kinase (FAK) subfamily and beta subtype protein 14-3-3 were up-regulated in the alpha-MHC Cre(+/-) ALK3(F/-) mice. These data provide support that ALK3 gene played an important role during heart development. The platelet-activating factors acetylhydrolase and Pax-8 genes could be important ALK3 downstream genes in the BMP signaling pathway during interventricular septum development. PTK and beta subtype protein 14-3-3 might be regulatory factors in this pathway. |
To express the GST-MAGE-3 protein in E. coli, and investigate the antitumor immune responses induced by Dendritic cells (DCs) pulsed with GST-MAGE-3 protein, the recombinant expression plasmid pGEX-MAGE-3 was constructed by ligating MAGE-3 gene, which was amplified by RT-PCR and confirmed by sequencing, and the pGEX-4T-2 vector. The recombinant plasmid was transformed into BL-21 E. coli. The expression of GST-MAGE-3 was induced with IPTG. The GST-MAGE-3 protein expressed as high as 32% of the total cellular protein. After purification with Glutathione Sepharose 4B, the purity of the protein was more than 90%, and 3mg GST-MAGE-3 was obtained from 100 mL BL-21 lysate. Dendritic cells from gastric carcinoma patients were pulsed with GST-MAGE-3 protein, and these DCs were used to stimulate the autologous T. lymphocytes. After 7 days, the T. lymphocytes cocultured with DCs pulsed with GST-MAGE-3 antigen exhibited specific cytotoxicity against MAGE-3 positive SGC-7901 cells. It is concluded that the GST-MAGE-3 protein are able to present antigen to T. lymphocytes, activate antigen-specific CTLs and induce special antitumor immune responses in vitro. Our results lay the groundwork for further research of the MAGE-3 vaccine. |
To obtain the expression of Mycobacterium tuberculosis heat shock protein 70 in methylotropic yeast. The expression vector pPIC9K-hsp70 was constructed, linearized and introduced into Pichia pastoris GS115 by electroporation. The result protein was secreted into the supernatant induced by 0.5% methanol at 30 degrees C and purified by centrifugation, ultrafiltration and ATP-agarose. The recombinant Hsp70 was identified by SDS-PAGE, Western blot, mice experiment and effect on the immature DC. The SDS-PAGE and Western blot analysis showed that the apparent molecular weight of expressed Hsp70 was about 70 kD and the expressed protein could specifically react with anti-Mt. Hsp70 IgG. And mice immunization indicated the expressed hsp70 had immunogenicity. Hsp70 could induce DC maturation and release Th1 cytokine. The secreted 70 kD protein was about 120 mg/L which accounted for more than 30% of the total supernatant protein and was purified to electrophoretic purity. The Hsp70, which had the biological activity, is successfully secretorily expressed in the Pichia pastoris GS115. |
Hydantoinase can be widely used in enzymic production of various amino acids. In order to obtain the hydantoinase genes in Arthrobacter BT801, its chromatosomal DNA is isolated and partialy digested with Sau3A I to collect fragments of about 30kb. Then, this fragment is inserted into the Hpa I and Pst I site of cosmid pKC505. The genomic library was thus constructed by packing in vitro with lambda phage package protein and transfecting E. coli DH5alpha. A positive transformant was selected from the library using thin layer chromatography and other methods. A DNA fragment containing complete hydantoinase genes was sequenced by sub-cloning into pUC18. The gene can express active protein under control of its own promoter and T5 promoter in E. coli. The isolation of the gene established foundition for research and application of the hydantoinase. |
In this article primary studies of the application of hyperbranched rolling cycle amplification (HRCA) in exogenous genes detection of transgenic plants were done. Four padlock probes were designed according to the sequences of four genes/DNA fragments that are used widely in transgenic plants; part of the sequence of pKK233 was chosen as the linking part of padlock probes and a pair of HRCA primers was designed according to the sequence of linking part. Study of the specificity of ligation in HRCA with isotope labeled padlock probes indicated padlock probes could be ringed effectively only when corresponding target DNA exited in the same reaction system and could not be ringed when there was no corresponding target DNA exited. Ligation time is very different according to the characteristic of target DNA being used. 5 min to 10 min is enough if the target DNA is plasmid; 30 min to 60 min is needed if the target is genome DNA of plant because it's sequence is more complex than that of plasmid's. HRCA time was analyzed which indicated longer reaction time can obviously increase the amount of products. Quantity of enzyme in HRCA was also analyzed. Different amount of enzyme (from 0.5 unit to 4 units) can give similar result when other conditions are not changed. On the basis of the research, transgenic tobacco was detected with these four padlock probes and the results were just as prospective. In order to increase the efficiency of detection, multiplex HRCA (MHRCA)was used. In MHRCA more than one padlock probes are used at the same time in the same reaction system to detect more than one targets. Because the amplification products of MHRCA will be complex and it is almost impossible to analyze with electrophoresis, so reverse-blot is used. Detection results of transgenic tobacco with this method are the same with anticipation. Compare to MPCR method we established before MHRCA is more convenient to operate and more effective in detecting exogenous genes in transgenic plants. |
The aim of this research is to prepare high quality polyclonal antibodies against Ecp, a recently identified leucine-zipper protein. The full length cDNA of ecp was amplified by PCR, cloned into pGEX-4T-1(His)6 and transformed into E. coli DH 5alpha. After induction with IPTG, the GST-Ecp fusion protein from the lysate was bound to glutathione-Sepharose 4B and digested with thrombin. The released Ecp protein was further purified through Ni-NTA affinity chromatography to homogeneity. A rabbit was immunized with the purified Ecp, and the antibody generated against Ecp was purified by affinity chromatography. The results of the Western blot showed that Ecp is present in various development stages of Drosophila melanogaster, from larvae to adult. |
beta-mannanase (EC 3.2.1.78) from Bacillus subtilis SA-22 was purified successively by ammonium sulfate precipitation, hydroxyapatite chromatography, Sephadex G-75 gel filtration and DEAE-52 anion-exchange chromatography. Through these steps, the enzyme was concentrated 30.75-fold with a recovery rate of 23.43%, with a specific activity of 34780.56 u/mg. Molecular weight of the enzyme was determined to be 38 kD by SDS-PAGE and 34 kD by gel filtration. The results revealed that the optimal pH value for the enzyme was 6.5 and the optimal temperature was 70 degrees C. The enzyme is stable between pH 5 to 10. The enzyme remained most of its activity after a treatment of 4 h at 50 degrees C, but lost 25% of activity at 60 degrees C for 4 h, lost 50% of activity at 70 degrees C for 3 h. The enzyme activity was strongly inhibited by Hg2+. The Michaelis constants (Km) were measured as 11.30 mg/mL for locust bean gum and 4.76 mg/mL for konjac powder, while Vmax for these two polysaccharides were 188.68 (micromol x mL(-1) x min(-1)) and 114.94 (micromol x mL(-1) x min(-1)), respectively. |
MUC1 mucin is a high molecular weight, type I transmembrane glycoprotein. High and aberrant expression of MUC1 is observed in various types of tumors, which make it an ideal target for tumor biotherapy as well as a biomarker for tumor diagnosis and prognosis. MUC1/Y is an isoform of MUC1 generated by alternative splicing. Specific expression of MUC1/Y in breast cancer as well as its involvement in tumor cell signal transduction have been reported. In order to purify peptides containing MUC1/Y-specific epitope in E. coli and prepare MUC1/Y-specific antibody, DNA fragment encoding the MUC1/Y-specific peptide was amplified by PCR using MUC1/Y full length cDNA as the template and cloned into fusion expression vector pGEX-2T, resulting pGEX-Y30. DNA sequencing was performed to confirm the correct amplification and orientation of the target sequence. Competent E. coli DH5alpha was transformed with pGEX-Y30 and the expression was induced for 4-5 hours in 0.2 mmol/L IPTG at 30 degrees C and 37 degrees C. Expressed proteins were released from the cells by ultrasonication or B-PER II reagent treatments. The fusion protein GST-Y30 were purified by affinity and anion exchange columns and identified by SDS-PAGE and Western-blotting. Polyclonal antibody was prepared by immunizing rabbits with the GST-Y30 protein for 4 times with intervals of 3 weeks and purified by GST column. Western blotting, ELISA and immunohistochemistry analysis were carried out using the purified antibody to confirm its MUC1/Y-binding capacity and specificity. The expressed fusion protein GST-Y30 is about 31 kD in size and represented about 20% of total cellular proteins. The majority of the GST-Y30 protein existed as soluble form when the induction was carried out at both 30 degrees C and 37 degrees C. After the two-step purification, the purity of GST-Y30 was about 94%. The titer of polyserum generated by GST-Y30 immunization was 1:320,000 by ELISA. The antiserum showed MUC1/Y specificity and can recognize MUC1/Y on MCF7 cell. The MUC1/Y-specific polyclonal antibody can be used for studying the role of MUC1/Y in carcinogenesis. |
Heavy metal wastewater poses a serious threat to the environment. In comparison to the existing methods of chemical precipitation, ion exchange and carbon adsorption, biosorption is an attractive alternative for the recovery of heavy metals from industrial effluents. However, nickel ion, different from other heavy metal ions, is a more recalcitrant pollutant and has low affinity to many metal tolerant microorganisms. In this study, Escherichia coli JM109 was genetically engineered to simultaneously express a Ni2+ transport system (the product of nixA gene) andoverexpress metallothionein (MT). NixA protein has a high affinity for Ni2+, and metallothioneins (MTs) are capable of binding a variety of heavy metals including Ni2+ . The Ni2+ bioaccumulation performance of the genetically engineered E. coli JM109 was evaluated. Time-course test showed that the bioaccumulation rate was rapid, and 95% of the accumulation was achieved within the first 10 minutes. The maximum Ni2+ bioaccumulation by genetically engineered E. coli cells was dramatically increased from 1.54 mg/g to 10.11mg/g, a more than five-fold increase than that of the original E. coli strain. The isotherm was of Langmuir type. Within the tested pH range (pH 4-10), the engineered cells displayed more resistance to pH variation, retaining up to 80% of the Ni2+ binding capacity at pH 4, while the original E. coli host cells lost 80% of Ni2+ binding capacity at pH 4. The presence of Na+ and Ca2+ affected Ni2+ bioaccumulation, but the effects were not serious, as 71% and 66% of the Ni2+ binding capacities were retained respectively at the concentrations of 1000 mg/L Na+ and 1000 mg/L Ca2+ . However, Mg2+ exerted a severe adverse effect on Ni2+ bioaccumulation, 83% of Ni2+ accumulating capacity was lost when Mg2+ concentration reached 200 mg/L. The effects of different kinds of heavy metals on Ni2+ accumulating were different. The genetically engineered E. coli cell lost less than 45% of its Ni2+ bioaccumulation activity in the presence of 50 mg/L lead or cadmium, 66% in the presence of 25mg/L mercury and 84% in the presence of 40 mg/L copper. The presence of glucose did not improve Ni2+ uptake. Our study suggests that the genetically engineered E. coli JM109 has potential application for effective and efficient recovery of nickel from aqueous solutions. |
Glutathione (L-gamma-glutamyl-L-cysteinylglycine), one of the major non-protein thiol compounds, is widely distributed in living cells and plays an important role in maintaining the normal redox environment of cells as an antioxidant. In the production of glutathione by fermentation, temperature is one of the most important environmental factors that affect the yield and the productivity of glutathione. Here the effect of temperature, varied from 24 degrees C to 32 degrees C, on the batch fermentation of glutathione in a 7 L stirred fermenter by Candida utilis WSH 02-08 was investigated. It was found that cell growth was hastened along with the increase of temperature. The maximum dry cell weight was achieved approximately 16 g/L under various temperatures, as soon as the glucose was exhausted. The effect of temperature on glutathione production was different from that on cell growth: the lower the temperature, the higher the glutathione production, i.e. the maximum glutathione concentration at 32 degrees C (235 mg/L) was only 75% and 64% of that at 30 degrees C and 26 degrees C, respectively. The maximum average specific growth rate (0.13 h(-1)) was achieved at 30 degrees C while the maximum glutathione concentration (366 mg x L(-1)) and the maximum intracellular glutathione content (2.3%) were obtained at 26 degrees C. Therefore, the optimum temperatures for cell growth and glutathione production are quite different in the batch fermentation. A modified Logistic equation was successfully applied to estimate the kinetics of cell growth. The maximum specific growth rate and the substrate inhibition constant, calculated from this equation, were both increased along with the temperature. In addition, the glutathione fermentation by C. utilis WSH 02-08 under various temperatures was proven to be a partial growth-associated process by estimating the process with the Luedeking-Piret equation. Based on the estimated parameon the estimated parameters, the effect of temperature on the kinetics of cell growth was further studied. An equation, dX / dt = [0.0224(T + 1.7)]2 X(1-X/Xmax) / 1 + S/ {8.26 x 10(6) x exp [-31477/R/(T+273)]}, was developed and applied to interlink the relationship between biomass concentration and temperature as well as substrate concentration in the batch glutathione fermentation. The experiment results showed that this model could predict the growth pattern very well. |
Northern corn leaf blight, caused by the fungus Exserohirum turcicum Pass. (Leonard and Suggs), is one of the major diseases in most corn-growing areas of the world. Research on gene tagging of E. turcicum has been limited due to the lack of an efficient transformation system. Since E. turcicum produces and accumulates melamin in cell walls during vegetative growth, it is difficult to efficiently isolate its protoplast. To isolate the protoplast of this pathogen with a high frequency, the effects of cell wall degradation enzymes, including beta-1,3-glucanase (Fungase, Funcelase, Novozyme and Glucanex) and beta-glucuronidase (Driselase, Uskizyme and Kitalase), enzyme concentrations, combinations, strains and medium on the isolation frequency were tested. The isolation frequencies were high enough for transformation when the combinations of (Kitalase + Glucanex + Driselase), (Kitalase + Glucanex) or (Kitalase + Uskizyme) were used. Moreover, the isolation frequencies of protoplast were significantly affected by the cultural morphologies of strain and the growth stage of mycelia. Among the plasmids tested, only plasmid pAN71 is efficient for transformation of E. turcicum. This result will provide some useful information for gene tagging of E. turcicum and other species in Exserohirum. |
A full-length cDNA encoding a MYB-related regulatory gene was isolated from a cDNA library prepared from mRNAs of the red line callus of S. medusa by TD-PCR. The cDNA, designated SmP, is 969 nucleotides long and has an open reading frame of 771 bp with a deduced amino acid sequence of 256 residues. The putative protein of SmP has two typical conversed R2R3-Myb DNA-binding domains in N-terminal and displays a rather high degree of similarity to OsMYB from rice and LBMI from tobacco, showing 73% and 70% identity within the DNA-binding domains. However, the C-terminal domain of the SmP protein does not show obvious similarity to any other known protein sequence. It is rich in hydrophilic amino acids, especially in serine residues (18.38%), partly organized in homopolymeric stretches, a feature often found in activation domain of transcription factors. |
In order to obtain the gene P12X3C of Foot-and-Mouth Disease Virus (FMDV) that includes full length P1, 2A, 3C and a part of 2B, the site mutation strategy was used. After being digested by Kpn I and Xba I respectively, the gene P12X3C was cloned into the pcDNA3.1 (+) expression vector. The recombinant plasmid was checked by restriction enzyme analysis and nucleic acid sequencing, and then named pcDNA3.1/P12X3C. Further, BHK-21 cells was transfected with pcDNA3.1/P12X3C by using lipoid. The proteins of Foot-and-Mouth Disease Virus, which were expressed in BHK-21 cells, were confirmed by sandwich-ELISA and fluoroscopy. The result shows the gene P12X3C is cloned into eukaryotic expression plasmid, and the recombinant eukaryotic expression plasmid pcDNA3.1/P12X3C could express proteins of Foot-and-Mouth Disease Virus in BHK-21 cells, which have immunocompetence. This study demonstrates that delivery of a recombinant eukaryotic expression plasmid containing P12X3C coding regions results in the assembly of FMDV capsid structures, which will offer experimental base to DNA vaccine of FMDV. |
The mechanism of oxidative refolding of proteins was elucidated in more detail from the intensive and extensive studies in the past decades. 1. Most of the proteins examined so far proceed oxidative refolding via multiple pathways rather than a single and specific pathway. This is consistent with the folding energy landscape theory. 2. It is the native interactions rather than the non-native interactions that direct the folding process. This is not necessarily incompatible with the importance of the non-native disulfide intermediates in the bovine pancreatic trypsin inhibitor (BPTI) pathway, which are just a chemical necessity in the intramolecular arrangement to facilitate native disulfide formation. 3. Based on the BPTI refolding it was suggested that disulfide bonds have a stabilizing effect on the native state without determining either the folding pathway or the final three-dimensional structure of the protein. This point of view is not applicable to other proteins. Studies on the refolding of prochymosin unequivocally demonstrated that the formation of native disulfides is the prerequisite to the recovery of the native conformation. It is more likely that the interdependence between the native disulfide formation and the formation of native structure is a general rule. 4. At the early stage of oxidative refolding disulfide formation is essentially a random process, with the progress of refolding further disulfide formation is increasingly dependent on the conformations of the intermediates. Enhancing the renaturation yield of recombinant proteins is a major challenge in biotechnology. In addition to aggregation, the formation of species with mispaired disulfide bonds is a leading cause of decreased yield. Progress in understanding the mechanism of oxidative refolding has provided insight into how to solve this problem. As described above, at the later stage of refolding disulfide formation depends on the conformations of intermediates. The intermediates with native-like and flexible structure favourable for native disulfide formation and correct refolding are productive intermediates, while the unproductive intermediates tend to adopt stable conformations, which render the thiol groups and disulfide bond(s) inaccessible and further folding unfavourable energetically. Therefore, the principle to enhance the renaturation yield of disulfide-containing proteins is to cause the productive intermediates to predominate by destabilizing the unproductive intermediates. To approach this, alkaline pH, low temperature, labilizing agents, protein disulfide isomerase and its analogues and alteration of primary structure have been proved useful to adjusting the structure of the unproductive intermediates so as to facilitate thiol/disulfide interchange and in turn the native disulfide formation. The prospects for the oxidative refolding of proteins both in basic and applied researches are discussed in this review article. |
Mice and 3-day-old chickens were orally inoculated with the recombinant attenuated Salmonella typhimurium strain ZJ111 carrying pcDNA3-F expression plasmid encoding the fusion protein of Newcastle disease virus (NDV). The results showed that ZJ111/pcDNA3-F was relatively safe. The recombinant plasmid pcDNA3-F was stable within the host stain ZJ111 in vitro and in vivo as shown by restriction enzyme analysis and PCR identification of the F gene. In an experimental vaccination study, 3-day-old chickens were orally immunized with ZJ111/pcDNA3-F with a dose of 108 cfu per chicken and boosted two weeks later. At week 4 post boosting, all chickens were challenged with a lethal dose of a virulent NDV strain F48 E9. The results showed that oral vaccination with ZJ111/pcDNA3-F induced stronger humoral and cellular immune responses than intramuscular immunization with naked pcDNA3-F plasmid. It also exhibited higher protection rate than the latter (66.7% vs 50%). This study indicates that the DNA vaccine using attenuated Salmonella typhimurium as delivery carrier had good safety, stability and immunogenicity and exhibited good potential of low cost and convenience for poultry disease control. |
Using the isolated total RNA from osteosacoma cell line MG63, the cDNA encoding human OPG was amplified by RT-PCR. A recombinant adenoviral vector carrying cDNA of OPG was constructed and OPG expression in mouse myoblast C2C12 cells was confirmed by Western blot and ELISA. The secreted expression of OPG protein persisted more than 6 weeks in vitro, and the growth of C2C12 cells infected by recombinant adenoviral were in good state. Osteoclasts derived from mouse bone marrow cells infected with recombinant adenoviral made less number of TRAP positive cells and resorption pits formed on dentine slices. |
Recently, we have reported a new gfp gene isolated from Aequorea macrodactyla. The protein purified from expressed E. coli exhibited an excitation peak at 476 nm and an emission peak at 496 nm. However, the drawback of only maturing to fluorescence at low temperature limited its applications. In this paper, we further describe twelve mutants of GFPxm. Seven mutants produced enhanced fluorescence when expressed in E. coli at higher temperature (37 degrees C). After six hours of induction at 25 degrees C, 32 degrees C and 37 degrees C respectively, the relative fluorescent intensities of GFPxm16, GFPxm18 and GFPxm19 were higher than that of EGFP, moreover GFPxm16 and GFPxm163 could preserve high fluorescent intensity even expressed at 42 degrees C. Four mutants of the seven could reach high expression level in three kind of mammalian cells. Another 6 mutants had red-shift of excitation-emission maxima, and longest excitation-emission maxima were 514nm and 525nm. Another three mutants had two excitation peaks, and one mutant had only one UV-excitation peak. The most exciting result is the mutant of OFPxm with orange color. The mutant has an excitation peak at 509 nm and an emission peak at 523nm. 523nm is yellowish green but the protein is orange observed by eyes. The mutant could reach high expression level and matured at higher temperature but the fluorescent intensity was comparatively low because of low quantum yield. |
A human membrane protein LASS2 (Homo sapiens longevity assurance homologue 2 of yeast LAG1), which has important physiologic functions, was expressed in three different expression systems. Only the LASS2 protein carboxyl terminal hydrophilic fragment could be expressed in the prokaryote expression system and its polyclonal antibody was produced. The full length of LASS2 protein could be expressed successfully in both eukaryotic in vitro translation system and Bacuvirus expression system: Bac-to-Bac system. SDS-PAGE analysis revealed that the molecular weight of expressed product of LASS2 was about 28 kD. The product was also proved by Western blot. This recombinant LASS2 protein was purified by metal affinity resin and the purity is above 90%. |
CpTI (Cowpea Trypsin Inhibitor) is a widely used insect resistance gene in the plant genetic engineering for its high insecticidal activity and the minimal ability of the insects to evolve resistance to it. To facilitate the safety assessment of genetically modified foods (GMFs) with CpTI protein, we need to produce gram quantities of this protein in microbes. With the pGEX fusion expression system, we expressed the GST-CpTI protein in E. coli BL21, which accounted for approximately 40% of germ proteins. By Glutathione Sephrose 4B affinity chromatography, GST-CpTI was obtained with the purity up to 90%. Overnight incubate the fusion proteins with Thrombin protease, we got the CpTI proteins cleavage of GST tag. Both of the GST-CpTI and CpTI proteins showed notable trypsin inhibitor activity. Immunization of rabbits with purified fusion protein generated high titer antibodies (> 20000), measuring by ELISA. Western Blotting also showed specific Ag-Ab binding band between the antiserum and the CpTI proteins no matter in the whole supersonic germ proteins or purified from the column. All these made a good ground for the further safety assessment of CpTI protein. |
To develop a fed-batch fementation process of E. coli TOP10 containing a recombinant plasmid pBAD/HBs Fab. Cells were grown in semi-defined medium at 37 degrees C, and the feed operation using glycerol as carbon source was performed when dissolved oxygen increased. When the target cell concentration reached to 64g/L, arabinose was added to a final concentration of 0.02%. Cells were grown for another 5h with the culture temperature decreased from 37 degrees C to 30 degrees C. In the whole process, cell growth was monitored by measuring OD600 of samples taken at 1/2h intervals and the dissolved oxygen was kept above 30%. After the fementation, E. coli pellets were collected for purification of Fab protein. The specificity of Fab protein was confirmed by Western blot, and binding activity to HBsAg was verified by Dot blot. Cell concentration we got is 96g wet bacteria per liter, the Fab protein is about 6% of total protein of the host, that is 80mg per liter. Stable fermentation parameters were obtained for fermentation to improve productivity of the Fab protein. The Fab protein was produced in the form of soluble biologically active protein, it's better than inclusion bodies from which biologically active protein can only be recovered by complicated and costly denaturation and refolding process. |
The permeability of cell membrane was enhanced by exogenous SA in the culture of Taxus cuspidata. Nuclei condense and fragments were observed in some cells by using fluorescent microscope, and the degraded chromosomal DNA was observed by using agarose gel electrophoresis. The changes in soluble proteins of the suspension cultures of Taxus cuspidata induced by salicylic acids were analyzed by using two-dimensional polyacrylamide gel electrophoresis. Comparing with the control, seven new protein spots were found and six protein spots were not found in the cultures grown with SA at 48h. The results obtained showed that SA could induce the expression of some special proteins that might be related with the action of SA in the suspension cultures of Taxus cuspidata. |
Biodiesel, an alternative diesel fuel, fatty acid alkyl ester, is made from renewable biological sources such as vegetable oils and animal fats. Two processes for biodiesel synthesis, enzymatic lipase catalytic esterification from fatty acid and transesterification from oils and fats, was investigated. The effects of various lipases, enzyme amount and purity, solvent, water absorbent, inhibition of short chains alcohol, specificity of substrate, molar ratio of substrate on esterification were studied in detail. The esterification degree with the optimal parameter and process can reach up to 92%. The purity of biodiesel obtained by separation and purification is up to 98%, and the half-life of the immobilized lipase for the esterification process can be up to 360hr, Moreover, the preliminary studies of the transesterification including the amount of methanol and mode of adding methanol into reaction system were made. The transesterification degree with adding methanol stepwise can reach 83%. |
The gene orf25 encodes functional protein that may play an important role in plant fertility control in nature. To clone the orf25 from Salicornia europaea Xinjiang into a T-vector, a single designed primer was used to amplify 1.7kb cDNA fragment with RT-PCR. Sequence analysis reveals that the cloned fragment contains entire orf25 coding region with 98%, 95%, 92% and 88% identity to that of orf25 from Beta vulgaris, Nicotiana, wheat and maize mitochondrion, respectively. This analysis suggests that orf25 gene is highly conserved in terms of evolution in plant; and it also suggests that wild plant Salicornia europaea contain a male-sterility gene similar to crops that is of great importance in improvement of the breed of crop. |
Human apolipoprotein A- I, the major protein component of high density lipoproteins and the main activator of the enzyme lecithin: cholesterol acyltransferase, defines the structure and stability and functions of HDL. It is clearly demonstrated that high concentration of the apoA- I not only inhibits the initiation and progression of atherosclerosis, but also makes the preexisting atherosclerotic lesions regress. This review gives an overview of the apoA- I structure, production, relation between apoA- I and HDL, and several mechanisms of the apoA-I anti-atherosclerosis. These mechanisms include directing excess celluar cholesterol from the peripheral tissues to the liver in reverse cholesterol transport, inhibiting oxidative modification of LDL, and modulating inflammatory responses to favour vasoprotection. |
The first bacterial hemoglobin(VHb) was found in a strictly aerobic bacterium, Vitreoscilla strain C1, occurring in marshes low in oxygen, but rich in organic matter. The hemoglobin gene is induced under low oxygen tension and may amount to 20 times as high. The expression of VHb promotes cell growth, protein biosynthesis and primary and secondary metabolism of the host cells, because the increased intracellular oxygen accelerates both the function of respiratory chain and terminal oxidases. The serial action of increased oxygen concentration is elucidated through yeast two hybrid system and a model is proposed. In addition, novel globin proteins known as flavohemoglobins have been isolated from various procaryotes and eucaryotes, with a N-terminal similar to VHb and C-terminal with reductase activity. Primary study shows that flavohemoglobin proteins exhibit similar function as VHb and also protection effect to nitrosative stress. Further work is needed to learn more about the physiology of these flavohemoglobins. The most remarkable physiological effects of VHb are exihibited in transgenic tobacco plants, including accelerated seed germination and growth in plant, increased synthesis of chlorophyll and dry weight. Without doubt, these effects are brought about through the increased oxygen supply to plant cells. It is deemed that VHb transgenic tobacco is a forerunner for transgenic crops and VHb may be a valuable route for staple seed crops. |
Genes encoding nucleocaspid (N) and membrane (M) protein of SARS coronavirus were obtained by RT-PCR and were cloned into expression vector pET22b and pBV222. DNA sequencing showed that the genes cloned from a patient in Beijing were identical to the gene sequences from reported Toronto strain. The genes were over-expressed in E. coli either as inclusion body or as soluble form. The recombinant proteins were purified by ion-exchange, or ion-exchange followed by metal chelate affinity chromatography. The recombinant N protein was demonstrated highly antigenic and could be employed as antigen to detect SARS antibodies in ELISA system for SARS diagnosis. |
The superantigen,such as staphylococcal enterotoxins, had been identified as possible anti-cancer molecules in many reports. In this paper, we cloned the entA gene encoding Staphylococcal enterotoxin A from the genomic DNA of Staphylococcus aureus(ATCC13565) by PCR, the sequence cloned was accordance with that reported in Genebank. The entA gene could be expressed effectively after inserted into plasmid pET-22b( + ), The rSEA was expressed as inclusion bodies when induced by IPTG at 37 degrees C and became soluble after induced at low temperature, the soluble part is about 55% of total rSEA products. Only one band was detected by western-blotting in expression product of BL-21 (DE3) with pET-SEA. The soluble rSEA was purified by Ni2+ chelating sepharose column. No other protein except rSEA was seen in SDS-PAGE gel stained by both Coomassie brilliant blue and silver salt, which showed that the rSEA was purified effectively. Homology modeling of rSEA determined the structure change was conducted, which indicated there was no apparent structure change between rSEA and native SEA. This result was also confirmed by proliferation assay of PBMC, for the rSEA could induced proliferation of PBMC as effectively as native SEA. The increasing anti-tumor activity of rSEA was also detected after the spleen cell activated in vivo by rSEA, which was accordance with others reports. This work paved the way for the further study of anti-cancer with rSEA. |
N-acyl-homoserine lactones (AHLs), are widely conserved signal molecules present in quorum-sensing systems of many Gram-negative bacteria. AHLs molecules mediate the expression of virulence genes of a range of bacterial pathogens. Recently, it has been reported that AiiA protein, which widely exists in Bacillus species, can inactivate the AHLs by hydrolyzing the lactone bond of AHLs, thus attenuate the diseases caused by the expression of virulence genes of bacterial pathogens. Bacillus thuringiensis, a type of Gram-positive bacteria, has been used extensively as a microbial insecticide in the last few decades. However, most of important insecticidal B. thuringiensis strains have not been exploited for bacterial disease control because they usually do not produce antibiotics that are effective against bacteria and fungi. The discovery of AiiA protein in B. thuringiensis shows the application potential of B. thuringiensis on biocontrol against bacterial diseases. In this study, in order to construct the B. thuringiensis recombinant strain that has high expression of AiiA protein, the promoter of insecticidal crystal protein coding gene cry3Aa of B. thuringiensis was selected. The promoter of gene cry3Aa is a non-sporulation promoter, it promotes the transcription earlier and longer than the promoters of other cry genes. The promoter of AiiA protein coding gene aiiA was replaced with the promoter of gene cry3Aa by overlapping PCR, resulting fusion gene pro3A-aiiA. The gene pro3A-aiiA was inserted into shuttle vector pHT304 at site BamH I / Sph I , resulting recombinant plasmid pBMB686. The plasmid pBMB686 was introduced into B. thuringiensis acrystalliferous strain BMB171, the resulting strain BMB686 had a higher and more stable expression level of protein AiiA comparing with the parental strain BMB171. Furthermore, the strain BMB686 exhibited stronger ability of AHLs inactivation and much more effective restraint to the potato's soft rot disease caused by Erwinia carotovora than those of the parental strain BMB171. From these results, it was concluded that the B. thuringiensis strain harvesting the fusion gene pro3A-aiiA may be utilized in the future to control bacterial diseases which are mediated by the AHL quorum-sensing signals. |
Streptomyces hygroscopicus 17997 produces the antiviral and antitumor ansamycin antibiotic, geldanamycin. Studies on geldanamycin biosynthetic pathway will provide good tools for genetic manipulation of the antibiotic-producing strain to improve the productivity or to facilitate making novel geldanamycin analogs. The structural similarities between geldanamycin and ansamycins such as rifamycin or ansatrienin suggest that both geldanamycin and ansamycins has a closely related pathways of biosynthesis and that biosynthetic system for geldanamycin is similar to the one of type I polyketide synthase (PKS) enzyme system. To explore the possible PKS genes involved in geldanamycin biosynthesis, the degenerate primers were designed according to the conserved sequence of KS-AT region from erythromycin and oleandomycin type I PKS genes. Cosmids containing multiple PKS genes (pCGBK2,4,6,10,11,18) were obtained by hybridization with the PCR products, which were amplified from S. hygroscopisus 17997 genomic DNA. The designed primers above were used for PCR. Development of a Streptomyces temperate phage phiC31-derivative KC515( tsrR) transduction system was carried out for identification of cosmids containing the PKS gene related to biosynthesis of geldanamycin. Several factors, mainly the Ca2+ and Mg + concentrations in different culture media affecting the frequency of gene transfection, were optimized .Transfection efficiency could reach up to 10(3) /microg DNA on YMG medium supplemented with 10mmol/L MgSO4. Reversely, the transfection efficiency decreased when YMG medium was supplemented with 30mmol/L MgSO4. Gene transfection system based on the integration-defective phage KC515 had been established for S. hygroscopicus17997. Recombinant phages (ph111, 258, 287, 116, 105) were constructed by insertion of the homologous to PKS gene fragments into the KC515 phage vector. Gene disruption experiments were performed by transduction of recombinant phages into S. hygroscopicus 17997 genome, and disruption of geldanamycin production was observed as a result of homologous recombination between the cloned insert in recombinant phage and the S. hygroscopicus 17997 genome by integration. Thiostrepton resistant transductants were selected and integration event was analyzed by Southern hybridization. The fermentation broth extracts from five resistant transductants were analyzed by TLC and HPLC. The results showed that only G16 mutant failed to produce geldanamycin. This result showed that the integration of the insert DNA fragment in recombinant phage phl6 into the chromosome of S. hygroscopicus disrupted the expression of the geldanamycin biosynthetic genes. The original cosmid pCGBK10 containing this cloned insert was predicted to encode PKS genes in the geldanamycin biosynthesis. This study laid the foundation for cloning the PKS genes involved in geldanamycin biosynthetic gene cluster from S. hygroscopicus 17997. |
Cysticercosis is caused by the metacestode form of Taenia solium-Cysticercus cellulosae and it causes great economic losses and threatens the people's health. There are some problems on how to control cysticercosis, in order to resolve the key problem that the native antigen to diagnose and prevent cysticercosis is very limited and is not satisfied, Pichia pastoris Expression System was used to express recombinant P2 protein. The interested P2 gene was got by digesting the pGEM - P2 vector using restriction endonuclease, then it was inserted into the secretory pPIC9K Pichia pastoris expression vector and transformed into E. coli. Positive recombinant plasmids were selected sequenced and named pPIC9K-P2 and it was linearized by Sal I and Bgl II, then the linear DNA transfored into Pichia pastoris GS115 by electroporation. The recombinant expression vector pPIC9K - P2 integrated into GS115 via homologous recombination between the transforming DNA and regions of homology within the genome. The transformants were screened for multicopy recombinants using G418 and were distinguished for Mut phenotypes by MD and MM. Two different phenotypes were generated-HIS+ MUT+ (Methanol utilization plus) and HIS+ MUT(S) (Methanol utilization slow). PCR analysis of the multicopy recombinants indicated that the P2 gene was integrated within the genome of pichia Pastoris. The multicopy recombinants were named GS115/pPIC9K - P2HIS+ MUT+ and GS115/pPIC9K-P2HIS+ MUT(S), both HIS+ MUT+ and HIS+ MUT(S) were induced with methanol. The results of SDS-PAGE and Western blot demonstrated that the culture supernatant of the induced Pichia pastoris contained P2 protein which was accumulated up to 33 % of total proteins in the culture supernant and its molecular weight is 12.6kD. The results of the clinical study indicated that the expression P2 protein could be used to diagnose human cysticercosis and swine cysticercosis as diagnosis antigen. |
Classical swine fever virus (CSFV), an enveloped positive-stranded RNA virus in the genus Pestivirus of the Flaviviridae family, is the causative agent of a highly contagious swine disease characterized by symptoms of hemorrhagic fever and immune depression, usually leading to substantial economic losses. The serological methods for detection of CSFV antibody such as ELISA are important means for the diagnosis of CSFV and immune surveillance. It is difficult to obtain CSFV antigen with high quality using traditional method because its titration titer is low in cell culture. CSFV has four structural protein named C, E0, El and E2. The E2 protein contains major antigenic determinants that are conserved between different CSFV strains and involved in neutralization by antibodies. So recombinant E2 protein can be developed as an alternative to the intact viral antigen. So far, CSFV E2 have not been expressed in E. coli with high level. Many factors, such as the secondary structure, the stability of 5' and 3' terminus of gene, the location of SD sequence and the bias of codes, are involved in the expressing level of foreign gene in E. coli . In this study, two sites of the E2 gene sequence were confirmed to be detrimental to its expression efficiency in E. coli through the computer-aided analysis. So they were mutated using recombinant PCR without changing the amino acids sequence of CSFV E2 gene. A plasmid was constructed by inserting the mutated E2 gene into the prokaryotic expression vector pET-28a(+) and named pETE2. The E. coli competent host BL21 (DE3)lysS transformed with pETE2 could express the E2 gene at high level, amounting to 28% of the total protein of the induced recombinant bacteria at the presence of IPTG. Except the hydrophobic transmembrane domain at C terminus, the recombinant E2 protein includes the total aa sequence. So it contains all the potential linear antigen epitopes of E2 protein because hydrophobic aa region can not form epitope. The recombinant E2 protein was CSFV-specific as proved by Western blotting and indirect ELISA. The rabbits immunized with the recombinant E2 can be protect from the challenge of hog cholera lapinized virus. This is the first report that E2 gene is expressed with high level expression in E. coli. In conclusion, it is an effective measure that mutate the CSFV E2 gene to increase its expression level in E. coli. The recombinant CSFV E2 protein possess fine immunonicity and can be used the antigen for the detection of CSFV antibody. |
The objective of this work was to develop a valuable adsorbent for recovery of platinum by studying the properties of Pt4+ -adsorption with immobilized Citrobacter freudii XP05 biomass. Five methods for immobilization of Citrobacter freudii XP05 biomass were compared. The method with gelatin-alginate sodium as entrapment matrix was considered to be the optimal. Spherical and uniform beads were produced and the SEM micrograph indicated that the cell of strain XP08 were uniformly dispersed within the matrix. The adsorption of Pt4+ by immobilized XP05 biomass was affected with adsorptive time, pH value of the solution, immobilized biomass concentration, Pt4+ initial concentration The adsorption was a rapid process. The optimal pH value for Pt4+ adsorption was 1.5, and its adsorptive capacity increased linearly with increasing Pt4+ initial concentrations in the range of 50 - 250 mg/L. The experimental data could be fitted to Langmuir and Freundlich models of adsorption isotherm. The adsorptive capacity reached 35.2 mg/g under the conditions of 250 Pt4+ mg/L, 2.0 g/L immobilized biomass, pH 1.5 and 30 degrees C for 60 min. 98.7% of Pt4+ adsorbed on immobilized biomass could be desorbed with 0.5 mol HC1/L. The characteristics of dynamic adsorption and desorption of immobilized XP05 biomass in packed-bed reactor were investigated. The saturation uptake was 24.66 mg Pt4+ /g under the conditions of flow rate 1.2 mL/min, pH 1.5, 50 mg Pt4+/L and 1.85 g biomass(dry weight) . Adsorptive efficiency of Pt4 + by the immobilized XP05 biomass was above 78% for 4 cycles of adsorption and desorption. The recovery of platinum from waste platinum catalyst was studied. The adsorptive capacity was 20.94 mg Pt4+/g immobilized biomass under the conditions of 4.0 g/L immobilized XP05 biomass, 117.76 mg Pt4+/L and pH 1.5 for 60 min. The immobilized XP05 biomass is potentially applicable to the recovery of platinum from waste and wastewater containing platinum. |
In recent years, the prevention and cure of infectious bursal disease (IBD) have become more and more difficult due to the emergence of very virulent strains of infectious bursal disease virus (vvIBDV) and the variant strains of IBDV. In this research, the hybridoma cell lines which secretes anti-idiotypic antibodies against anti-IBDV IgG were established. According to the Jerne's theory of immune network, the use of the anti-idiotypic antibodies as a vaccine will be a new method for the prevention of IBD. In this study, the SPF chickens were inoculated with the IBDV- SD strain, and the bursal was obtained from the died chickens. The bursal was then homogenized and frozen-thawed 3 cycles, and the virus samples were prepared by cane sugar density gradient centrifugation and dialysis. Typical IBDV particles were observed under an electron microscope, and the concentration of the virus protein measured by ultraviolet absorbance spectrophotometry was 10.8 mg/mL. SPF chickens were immunized with the virus and the highly immunized sera were prepared and purified by Sulfuric acid ammonia salt out and Sephadex G-25 chromatography. Then, Balb/C mice of six or eight weeks old were immunized interapertoneally(I. P.) with purified antibodies to IBDV at regular intervals. SP2/0 myeloma cells were fused with the spleencytes from the immunized mice at a ratio of 10:1, in 50% polyethylene glycol (1540) and were then cultured in HAT until all the SP2/0 cells died. The hybridoma cells were selected by ELISA and the highly positive holes were cloned 3 times with the method of limited dilution. Two strains (2B6 strain,5F4 strain) of hybridoma cells were obtained, which were shown by ELISA to steadily secrete anti-IBDV idiotypic antibodies. The chromosome number of the two hybridoma cells were about 88 - 106, 95 in average, and the antibodies secreted belonged to the types of IgG1 and Kappa. Balb/c mice of 3 months old were inoculated I.P. with about 10(7) hybridoma cells per capita, and the ascites were collected 12 days later and the titre of anti-IBDV idiotypic antibodies measured by ELISA was 1 :25600 (for 2B6) and 1:12800 (for 5F4) . The ascites containing the anti-IBDV idiotypic antibodies were emulsified with complete or incomplete Freund's adjuvants, and the anti-IBDV idiotypic antibody vaccine was obtained. SPF and common Jingbai chickens were immunized with the vaccine obtained. The immunized chickens with the vaccine were inoculated with IBDV-SD strain at a dose of 2000 ELD50 after twoimmunizations. All the 10 SPF chickens in the non-immunized group were sick, and 8 of them died; and 5 out of the 50 SPF chickens immunized group got sick and 2 died. All the 10 common Jingbai chickens in the control group were sick, and 6 died; 7 of the 30 immunized common Jingbai chickens got sick and only 1 died. Chi2 analysis showed that the difference between the immunized and the non-immunized groups in both the SPF and the common Jingbai chickens were significant (P < 0.01). Our result indicated that the anti-IBDV idiotypic antibody vaccine well protected chickens and had a great potential in both research and clinical application. |
To clone cDNA of human leptin gene and obtain leptin protein for future study on leptin binding proteins. The cDNA of human leptin with 6 x his-tag was cloned by over-hang extension PCR protocol using human genomic DNA as template, and subcloned into in vitro expression vector pIVEX2.3MCS, and the fusion protein was expressed in vitro by Rapid Translation System (RTS) (RTS500 cycle primer Kit and RTS500 ProteoMaster of Roche company). The apparent molecular weight(19.46 kD) and the immuno-specificity of the fusion protein were confirmed by SDS-PAGE and Western blot, and the expressed fusion protein stayed mainly in the supernatant of the reaction mixture in soluble form. This work provides us solid basis for further study on new leptin-associated proteins. |
The objective of this study was to develop research of cardiac cells to reestablish 3D tissue architecture in vitro, we performed studies using collagen membrane as three-dimensional scaffold for cardiac cells culture with the principles and methods of tissue engineering. The polymer scaffold provides a 3-D substrate for cell attachment and tissue formation. Cardiac cells isolated by enzymatic digestion from 1d old neonatal rats were seeded to three-dimensional collagen scaffolds and tissue culture plates. The morphology, beating rate and the metabolic indexes, including specific consumption rate of glucose (q(glu)) , specific production rate of lactate (q(lac)), lactate transform rate ( Y(lac/glu)), specific creatine kinase (CK) and lactate dehydrogenase (LDH) activities of cardiac cells cultured on three-dimensional collagen membrane and tissue culture plates were compared. It was found that cells shape and cells' CK and LDH activity was no differences between 3D and 2D cultures and cell beat rate on cell culture cluster was slower than those cells cultured on collagen membrane, However the cell glucose consumption and lactate yield rate of cells cultured on cluster was higher than those cells cultured on collagen membrane. After 5 days of cultivation, cardiac cells cultured on collagen membrane scaffolds organized into three-dimensional (3D) aggregates as opposed to the two-dimensional (2D) aggregates mosaic pattern seen in tissue culture plates, and spontaneous and rhythmical contractile 3D cultures in unison were visible to the naked eye and the area of synchronous contract three-dimensional (3D) aggregates reaches 80cm2. The mean value of q(glu), q(lac) and Y(lac/glu) of cultured on three-dimensional collagen scaffold was 7.37 micromol/10(6) cells/d, 2.92 micromol/10(6) cells/ d and 0.38 micromol/micromol, versus 7.59 micromol/10(6)cells/d, 3.83 micromol/10(6) cells/d and 0.51 micromol/micromol in tissue culture plates. These results demonstrate that cardiac cells immobilized on collagen membrane in 3D cultures maintain similar metabolic activity and contractile function when compared with native cardiac cells. The above results support the idea that engineered cardiac tissue can be used as a model of native tissue for studies of tissue development and function in vitro and eventually for tissue repair in vivo. |
The HIV-1 capsid protein (CA) plays an essential role in viral core assembly and maturation. Proteolytic cleavage at the MA-CA junction of the retroviral gag polyprotein refolds the amino-terminal end of capsid into a beta-helix structure that is stabilized by a salt bridge between the protein's processed amino-terminus and a conserved acidic residue. The refolded capsid aminoterminus then creates a new CA-CA interface, allowing assembly of the mature capsid core. Recently, researches focus on assembly of CA in vitro and development of CA vaccine. CA vaccine will provide widely immune protection because CA is comparatively conserved. Experiments demonstrate that fusing as few as four matrix residues onto the amino-terminus of capsid redirects protein assembly from cylinder to spheres in vitro. Evaluation of immunogenicity showed that immunization with virus-like particles induced both cellular and neutralizing antibody responses. Furthermore, mucosal administration of virus-like particles effectively induced both mucosal and systemic immune responses. These results indicate that virus-like particles consisting of HIV structural proteins are an attractive vaccine platform for eliciting anti-viral immune responses, especially neutralizing antibody responses. The production of antigens for vaccines in plants indicates that plant-based transgenic expression represents a viable means of producing CA vaccine for the development of HIV vaccine and for use in HIV diagnostic procedures and it has the potential as a safe and cost-effective alternative to traditional production systems. |
To construct a vector for DNA vaccine and protein expression by using chromosome-plasmid balanced lethal system which was based on the thyA+ gene/deltathyA Escherichia coli. The thyA genes from Escherichia coli and Vibrio cholerae were amplified by polymerase chain reaction and cloned into pCDNA3 by replacing ampilicilin resistant gene. Multiple cloning sites, the prokaryotic replicon, CMV promoter and the boving growth hormone polyA signal were also included in the vectors. Two new non-antibiotic recombinant plasmids renamed as pcDNATE and pcDNATC which had the nutritional marker as thyA were constructed and were transformed respectively into the deltathyA derivative of E. coli K-12 strain DY330-TI, then two chromosome-plasmid balanced systems for E. coli based on the thyA were developed. To test the efficiency and stability of the newly constructed chromosome-plasmid balanced lethal system, a reporter gene--red fluorescent protein (DsRed2) gene was cloned into pcDNATE, pcDNATC and expressed as fusion to the c-myc. The two recombinant plasmids, pcDNATE-DsRed2, pcDNATC-DsRed2, were transfected into HEK293 solely and DsRed2-myc was detected by the fluorescence microscope assay and western-blot. Meanwhile, the loss of recombinant plasmids were not seen in cultures without thymidine after 20 generations. The chromosomal-plasmid balanced lethal system is proved to be an effective vector system for the expression of target genes and share the same stability with the antibiotic-resistant plasmid vector system. It holds great potential in gene vaccine vector because obviating the weakpoints of the drug resistance marker during application. |
Heat-labile enterotoxin (LT) from Escherichia coli is a bacterial protein toxin with an AB5 hexamer structure. LT is a powerful mucosal adjuvant when co-administered with soluble antigens. However, its use in mucosal immunity is inconvenient because of its low yield and depolymerization during long-term storage under normal condition. In this study, we report an efficient expression system and optimized purification and storage strategy of LT. A gene encoding LT was cloned into the vector pET11c and transformed in E. coli BL21(DE3). By growing this strain on modified M9-CAA medium, LT was expressed efficiently. About 46mg/L LT could be purified from the supernatant of bacteria lysate. Using D(+)-Immobilized galactose column, LT could be purified at a wide pH range with various elution buffers. The optimized elution buffers are TEAN (pH 7.3) containing 0.3mol/L galactose and carbonate buffer (pH 10.4) containing 0.3mol/L galactose. After dried by freeze and placed in 4 degrees C, LT dissolved in TEAN (pH 7.3) and carbonate buffer (pH 10.4) were assayed by HPLC. The results indicated that the integrity of AB5 hexamer was kept well. LT could undergo long-term storage under this condition. This was proved to be an optimized strategy of LT storage. The results of GM1 binding assay and toxicity assay showed that the purified recombinant LT has normal biological character. |
Vip3A, a novel insecticidal protein, is secreted by Bacillus thuringiensis (Bt) during vegetative growth. Vip3A protein possesses insecticidal activity against a wild spectrum of lepidopteran insect larvae. Since the first cloning of vip3A gene from Bt, many other vip3A genes have been isolated. To investigate vip3A genes contribution to Bt and reflect the revolution relationships, the strains containing vip3A genes were screened and gene similarity was analyzed. 114 wild-type Bacillus thuringiensis (Bt) strains isolated from different regions and 41 standard Bt strains from the Institute of Pasteur were screened for the vip3A genes using PCR amplification. 39 strains including B. thuringiensis subsp. kurstaki (Btk) HD-1 were found to contain the vip3A genes. Because acrystallerous strain Cry- B derived from Btk HD-1 was proved not to contain vip3A gene, it suppose that the vip3A gene may be located at the plasmids. Vip3A proteins expressed in these strains were detected with polyclonal antibody by Western blot and 4 strains among them were shown not to express the Vip3A proteins. The vip3A genes amplified from wild-type Bacillus thuringiensis strains S101 and 611 with different levels of activity against lepidopteran insect larvae were cloned into pGEM-T Easy vector. Alignment of these 2 putative Vip3A proteins with 6 others (Vip3A (a), Vip3A(b), Vip3A-S, Vip3A-S184, Vip83 and Vip3V) in the GenBank data base and 2 reported Vip3A proteins (Vip14 and Vip15) showed that vip3A genes are highly conservative. The plasmids pOTP-S101 and pOTP-611 were constructed by in- serting 2 vip3A genes (vip3A-S101 and vip3A-611) into the expression vector pQE30 respectively and were transformed into E. coli M15. E. coli M15 cells harboring the pOTP plasmids were induced with 1 mmol/L IPTG to express 89 kDa protein. Experiments showed that the level of soluble proteins of Vip3A-S101 in E. coli M15[pOTP-S101] and Vip3A-611 in E. coli M15 [pOTP-611] were about 48% and 35% respectively. Bioassay showed that each of these Vip3A proteins had similar toxicity against neonate Spodoptera litura larvae, indicating that some amino acids change had little effect on the insecticidal activity of proteins. Although vip3A genes are conservative, the unknown insecticidal spectrum is still to be brought out. Vip3A genes can be used for the construction of the Bt engineered strains and transgenic plants. In addition, vip3A genes are excellent candidates for delay of the pest resistance due to the difference of the action model from that of Bt delta-endotoxins. |
To purify the recombinant human BMP-6 protein and to establish its in vitro bioassay method. The cDNA encoding the mature peptide of hBMP-6 protein was amplified by reverse transcription-polymerase chain reaction (RT-PCR), using human placental mRNA as template, and subcloned into the high-expression vector pET-15b under the control of T7 lac promoter. The resulting construct, pET-BMP6, was then transformed into an Escherichia coli strain BL21 (DE3) for the production of recombinant hBMP-6 protein (rhBMP-6). After 4 hours of induction by isopropyl-beta-D-thiogalactoside (IPTG), rhBMP-6 (approximately 15kD) was expressed and formed inclusion bodies, contributing up to 10% of the total bacterial protein. The inclusion bodies were isolated and redissolved in 8mol/L urea, and the denatured rhBMP-6 was purified to 95% purity by CM-Cellulose 32 ion exchange chromatography (IEC). The osteoinductivity of rhBMP-6 was measured by the expression of some of the osteoblast differentiation marker genes in rhBMP-6-treated C3H10T1/2 cells as reflected by determinations of alkaline phosphatase (ALPase) activity and semi-quantitative RT-PCR. At the end of the purification process, about 80% of rhBMP-6 formed disulphide-linked homodimers after refolding during renaturation. The apparent size of the protein was 30kD on non-reducing SDS-PAGE, similar to that of the native form of hBMP-6. The enzyme assays showed that the ALPase activity was increased in a dose-dependent manner with the treatment of rhBMP-6. After the addition of 300ng/mL of rhBMP-6, the ALPase activity of C3H10T1/2 cells increased significantly. The activity of rhBMP-6 used was comparable to about 70% of that of the standard hBMP-6 derived from eukaryotic cells. RNA extraction data also showed rhBMP-6 stimulated expression of osteoblast marker genes, including type I collagen, osterix, and osteocalcin in a time-dependent manner. After 5 days of treatment, their level of expression was increased to 3 times that of controls. Bone morphogenetic protein (BMP)-6, a member of the 60A subgroup of the bone morphogenetic protein (BMPs) family, plays a pivotal role in bone formation. Previous evidence showed that BMP-6 is selectively up-regulated by estrogen, suggesting its potential role in the treatment of osteoporotic fractures, especially for menopausal osteoporosis. Our present study demonstrates that the recombinant hBMP-6 produced in Escherichia coli is able to induce pre-osteoblastic cells to differentiate into osteoblasts in vitro, and analysis of mRNA expression of type I collagen, osterix, and osteocalcin can be also a method for measuring the osteoinductivity of BMP. This provides the basis for further studies on ectopic bone formation in the body and for the development of auxiliary drugs for the treatment of osteoporotic fractures. |
The uracil in DNA comes from either the misincorporation of dUTP in place of dTTP or deamination of cytosine. In the latter case, it can result in a GC to AT transition mutation if the uracil is not removed before DNA replication. Base excision repair (BER) is a major pathway for removing DNA lesions arising from endogenous processes as well as those induced by exposure to exogenous chemicals or irradiation. BER is initiated by DNA glycosylases that excise aberrant bases from DNA by cleavage of the N-glycosidic bond linking to the base of its deoxyribose sugar. Uracil N-glycosylase (UNG) is the enzyme responsible for the first step in the BER pathway that specifically removes uracil from DNA. The UNG gene undergoes both temporal and spatial regulation mainly at the level of transcription. Normally cancer cells undergo over-proliferation and up-regulate their UNG during tumorigenesis. In this study we examine the correlation between UNG level and carcinogenesis, and explore the possibility of using UNG as a marker for cancer diagnosis. Human UNG gene was amplified from the total RNA of the human choriocarcinoma cell line, JEG-3, by RT-PCR. After purification, the 942bp full-length UNG cDNA coding sequence was digested with EcoR I and Sal I, and cloned into the digested pET-21 to construct a recombinant vector, pUNG. The UNG protein was expressed under the control of T7 promoter in E. coli BL21 (DE3) cells induced with IPTG. After ultrasonic treatment, the cell lysate and precipitate were analyzed by SDS-PAGE and a 39kD band was detected. The plasmid was serially diluted at appropriate concentrations and employed as standards in the subsequent quantification. Total RNAs were extracted from 18 pairs of clinical samples, each pair contains a sample of esophageal squamous cell carcinoma (ESCC) tissue and its surrounding normal esophageal epithelia. The copy numbers of UNG mRNA in these RNA samples were determined by real-time quantitative RT-PCR using a Lightcycler (Roche). UNG was present in 13 cases of ESCC (13/18, n = 18) but absent in all of the normal tissues. The results indicated that there was a correlation between high level of UNG expression and the carcinogenesis of ESCC. |
The Cry1Ab differs most significantly from the other related ICPs by its absence of a carboxyl terminus of 28 amino acids including four cysteines; consequently it is less stable. We report that the helper protein P20 plays a role in the expression and crystallization of Cry1Ab. Three Cry1Ab expression plasmids pT1B, pP1B, and pDP1B, were constructed based on the shuttle vector pHT3101. The vector pT1B does not contain the p20 gene, pP1B carries p20, and pDP1B contains p20 with cry1A(c) promoter. Transformants were obtained by electroporating the plasmids into Bacillus thuringiensis acrystalliferous mutant CryB. Western blot demonstrated that crylAb was expressed as a 130 kD protein in all the transformants, and some of the protein was partially degraded into a 60 kD peptide. Quantitative protein analysis indicated that the amount of the 130 kD protein varied in the transformants and was in the ratio of 1:1.4:1.5 for PT1B, pP1B and pDP1B respectively. For the 60 kD proteins, the ratio was 1:1.1:1.6. Microscopic examination revealed that the size of the typical pyramidal crystals in the three transformants was in the order of T1B < P1B < DP1B. Bioassay showed that T1B, P1B and DP1B were all toxic to the larvae of Helicoverpa armigera with similar LC50. This study suggested that P20 plays a role in the expression and crystallization of Cry1Ab. |
A new method for manufacturing three-dimensional gel film-coated chips was described in this paper and its advantages were evaluated by its application. A patch of polyacrylamide gel (15mm x 15mm x 20 microm) was fixed on the glass surface with Bind-Silane treatment, then activated by glutaraldehyde. The aldehyde groups in gel provided reactive sites that allowed covalent immobilization of molecules containing amino groups. Oligonucleotides were mechanically spotted by GMS 417 Arrayer. After hybridization with Cy-3 labeled probes, fluorescence signals of perfect binding can be discriminated from mismatched ones. Compared with two-dimensional glass chip, the capacity of oligonucleotides immobilized on gel film-coated chip is over 100 times. And the gel film-coated chip have lower background and shorter hybridization time. Monoclonal antibodys of cytokine IL-4, IL-5, IL-6, IL-7, ANG, I-309 and VEGF were also immobilized on the gel film-coated chips to make protein microarrays. After incubation with serum of breast cancer patients or normal persons, the microarray reacted with biotin-labeled second antibodys of cytokines and Cy-3-labeled streptavidin sequentially. Results show IL-4, IL-5, I-309 and VEGF of patients have higher expression level than normal persons. This kind of protein microarrays can be potentially helpful to clinical diagnosis. Furthermore different oligonucleotides or proteins can be performed in parallel in a single reaction with minimal amount of binding reagents. Such gel film-coated chips can be used widely in the fabrication of oligonucleotides and proteins microarrays. |
Aflatoxins, found in contaminated food, are potent hepatocarcinogen. The aflatoxin-detoxiczyme (ADTZ) isolated from the edible fungus Armillariella sp., detoxifies aflatoxin B1 (AFB1). This paper reports on the characterization of immobilized ADTZ using a hydrophobic adsorption method. The ADTZ was isolated from cryo-homogenated fungus, previously cultivated at 24 - 28 degrees C for 20 - 30 days, using n-alkyl amino-agar beads. Various adsorption conditions of the enzyme to n-alkyl or n-octyl amino-agar beads were carried out. The effects of enzyme immobilization on different alkyl amino-agar beads, at different pH values (5.5 - 7.5), at different temperature (20 - 40 degrees C) and at different salt concentrations were investigated. The enzyme activity was measured at OD360 by reacting 133.3 ng/mL of AFB1 at 30 degrees C for 30 min with the immobilized ADTZ. The Km value of the immobilized enzyme, determined using Schematic Linewearver-Burk plot, is 3.308 x 10(-3) mol/L, lower than that of free enzyme, which is 2.16 x 10(-6) mol/L. This indicated the affinity of the detoxiczyme to AFB1 decreased after immobilization. The immobilized enzyme activity in oil-phase (n-hexane) was also studied with different concentration of water. After the treatment of the immobilized ADTZ, the toxin no longer causes liver toxicity in the rat toxicity test, no longer causes mutagenicity in Ames test and is no longer toxic in the chicken embryo test. Results also indicated that the pH stability, the thermostability and the freezing stability of ADTZ were improved after the immobilization. |
Chemostat culture was performed to characterize the growth, substrate consumption and the hirudin production, and to disclose their interrelations in the fermentation of recombinant Pichia pastoris. The Andrew substrate-inhibited growth model is more suitable than Monod model to simulate the growth of Pichia pastoris on methanol. Therefore, two stationary states can be obtained in the continuous culture at a certain dilution rate because of the substrate inhibition on cell growth. The stationary state could be obtained if only the dilution rate not more than 0.048 h(-1) in the continuous fermentation. The concentrations of cell, methanol and hirudin were constant after 50 h continuous culture with dilution rate at 0.04 h(-1). However, it could not be obtained when the dilution rate more than 0.048 h(-1) because the other stationary point at S > 0.048 h(-1) is unstable. Therefore, it was found that the cell concentration declined and the methanol concentration increased from 2.9 g/L to 18.1 g/L within 18h at dilution rate 0.06 h(-1). Thus, the fed-batch culture with a constant specific growth rate was carried out to disclose the fermentation behavior at high and constant methanol concentration in aid of a methanol sensor. The theoretical maximum specific growth rate, microm = 0.0464 h(-1), was found under critical methanol concentration, Scrit = 3.1 g/L. The growth of P. pastoris was typically methanol-limited at the methanol concentration S < Scrit. It was, however, inhibited at S > Scrit. The maximum specific Hir65 production rate qp was obtained at 0.2 mg/(g x h) when methanol concentration and mu were 0.5 g/L and 0.02 h(-1), respectively. The specific Hir65 production rate qp increased with the increase of mu and S at mu < 0.02 h(-1), and decreased at mu > 0.02 h(-1). The specific methanol consumption rate increased with the increase of S when S < 5 g/L, but decreased when S > 5 g/L. At last, the high Hir65 production rate 0.2 mg/(g x h) was obtained in the fermentation conducted under methanol-limited concentration and mu controlled at 0.5 g/L and 0.02 h(-1), respectively, while the specific methanol consumption rate is low only at 0.04 g/(g x h), showing the potential for the strategy of getting high Hir65 production rate at the low consumption of methanol. |
The United Nations Environment Program estimates that approximately 20% of agricultural land and 50% of cropland in the world is salt-stressed. The gene NHX (Na+/H+ exchanger) encodes functional protein that catalyzes the countertransport of Na+ and H+ across membranes and may play an important role in plant salt tolerance. To clone the NHX from the wild plant Populus euphratica collected in Tarim basin and Xinjiang Wujiaqu district into a T-vector, designed primer was used to amplify 1kb NHX cDNA fragment with RT-PCR. Total RNA was extracted from Populus euphratica tissue (plant tissue was collected from Tarim basin and Xinjiang Wujiaqu district and stored in liquid nitrogen) according to the Plant RNA Mini Kits of Omega. First cDNAs were synthesized from 1 microg total RNA of Populus euphratica seedling. A pair of primers were used to perform RT-PCR. The amplified DNA fragment was purified and cloned into pMD18-T vector. However, 1kb and 2.3kb fragment were obtained from Tarim basin and Xinjiang Wujiaqu district and named as PtNHX and PwNHX, respectively. Sequence analysis reveals that the cloned PtNHX fragment of Populus euphratica contains partial NHX coding region with 98%, 86%, 84% and 80% identity comparing with Atriplex gemelini, Suaeda maritima, Arabidopsis thaliana and Oryza sativa, respectively. This analysis suggests that NHX gene would be highly conserved in terms of evolution in plant; and it also suggests that the NHX gene of Populus euphratica also would have the similarity with that of Arabidopsis. It may be of great importance in improvement of the plant salt tolerance and breed of crop. At the same time, sequence analysis shows that PwNHX gene includes a coding region about 1350bp with 99% identity comparing with transposon Tn10 IS10-left transposase of Shigella flexneri. On the one hand, the NHX gene may lose its function because it was inserted a fragment in coding region. On the other hand, its product may play a important role in salt tolerance. Populus grow in saline soil. It speculates that it may have other salt tolerance mechanism in Populus. The transposon can be used as transposon tagging to clone other genes and it will help us to understand farther the salt tolerance mechanism. |
Excessive nitrate accumulated in plants affects vegetable quality severely and excessive nitrate ingestion would do harm to human health. Assimilatory NADH: nitrate reductase (NR, EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)- and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. Enhancing the activity of NR is conducive to reduce the concentration of nitrate in plants. The experiments were conducted to investigate the activity of nitrate reductase in different plant tissues and the relationship between external inducing solution concentration and NR activity (NRA) in plant leaves. Six plant seedlings growing in solution culture were deprived of an external nitrogen (N) supply for 2 weeks. On selected days, three of six plant seedlings were exposed to 50mmol/L NO3- for 0, 2, 5, 8, 11h, and four of the six plant seedlings were exposed to 0, 10, 30, 50mmol/L NO3- for 2h. The NRA was determined in vivo at 538nm using spectrophotometer. The results showed that NRA increased when those plant seedlings were induced by nitrate solution. The change trends of NRA in roots and in leaves of cole, pea and tomato were different during treating time. The NRA in cole leaves was higher than that in its root and in other two plants and increased along with inducing time, but the NRA in bea and tomato was highest when the treating time was 8h and 2h, respectively. The highest NRA in leaves of three kinds of Chinese cabbages and tomato was induced by different concentrations of KNO3 solution. In tomato leaves, the highest NRA was induced by 10 - 30mmol/L KNO3 solution. In three Chinese cabbages, Brassica chinensis L. cv. AJH, XBC and KR-605, the highest NRA was induced by 10, 30, 10mmol/L KNO3 solution, respectively. The results indicated that the response manners of NRA in plants to external nitrate solutions were different. According to these results, the level of NR mRNA in plants could be enhanced by nitrate inducement. The total RNA was isolated from tomato leaves and root which induced by 30mmol/L KNO3 solution for 2h, and NR cDNA was obtained by RT-PCR using the specific primers. The fragments of PCR products were cloned and sequenced. There are 2736 base pairs in the whole cDNA fragment. The deduced protein sequence contains 911 amino acids. The NR gene can be fused to the CaMV 35S promoter, then introduced to higher plants, such as vegetables. It is hoped to decrease drastically the nitrate content of the transgenic plants. |
Pathway engineering was the third generation of gene engineering. Its main goals were to change metabolic flux and open a new metabolic pathway in organism. Application of recombinant DNA methods to restructure metabolic networks can improve production of metabolite and protein products by altering pathway distributions and rates. Ethanol is the most advanced liquid fuel because it is environmentally friendly. Enhancing fuel ethanol production will require developing lower-cost feedstock, and only lignocellulosic feedstock is available in sufficient quantities to substitute for corn starch. Xylose is the major pentose found in lignocellulosic materials and after glucose the most abundant sugar available in nature. Recently a lot of attentions have been focused on designing metabolic pathway of Saccharomyces cerevisiae in order to expand the substrate of ethanol fermentation, because it is a traditional ethanol producing strain and has wonderful properties for ethanol industry. However, it can not utilize xylose but convert the isomer, xylulose. Many attempts are based on introducing the genes in the pathway of xylose metabolism. The further research includes overexpressing the key enzyme or decreasing the unimportant flux. The sugars in lignocellulose hydrolyzates, therefore, could be efficiently utilized. Here, we describe the ethanol pathway engineering progress in ethanol fermentation from xylose with recombinant Saccharomyces cerevisiae. |
Endoscopic injection sclerotherapy (EIS) is conducted to treat esophageal varices associated with liver cirrhosis. Cirrhotic patients have protein energy malnutrition (PEM), and their malnutrition is aggravated during EIS due to dietary restrictions. The aim of this study was to evaluate the dietetic benefits of oral branched chain amino acid-rich nutrient (BCAA-rich nutrient) administered during EIS. Cirrhotic patients admitted for prophylactic EIS were divided into three groups: Group A given a general liquid nutrient in two divided doses, Group B given a BCAA-rich nutrient in two divided doses, and Group C given the BCAA-rich nutrient in four divided doses. The nutritional state was evaluated based on physical examinations, biochemical parameters, and indirect calorimetry. A total of 29 patients were enrolled. Blood biochemical examinations showed a significant increase in the Fischer's ratio in Groups B and C. Group B showed significant decreases in resting energy expenditure/basal metabolic rate, while Group C showed a significant rise in non-protein respiratory quotient. The oxidation rate of carbohydrate significantly increased and that of fat significantly decreased in Group C. Administration of the BCAA-rich nutrient is considered to be useful in improving abnormalities of energy metabolism during EIS. |
Major surface protein (p30) and Dense Granule Antigen GRA6 of Toxoplasma gondii have good antigenicity, and could be used for detection of IgM against Toxoplasma gondii. GRA6 may complement P30 to reach more high sensitivity for detection of antibodies to Toxoplasma gondii, so, we try to express the chimeric protein of GRA6 and P30 by genetic engineering, identify its antignenicity and use for developing diagnosis reagent. Antigenic domains of p30 and GRA6 of Toxoplasma gondii were screened by analyzing their sequences using the software ANTHEWIN. Two DNA fragments encoding respectively antigenic domains of p30 and GRA6 were cloned, they were inserted into the same expression vector pET28a( + ) and expressed as a chimeric protein in Escherichia coli. BL21(DE3), the expressed chimeric protein of p30 with GRA6 in a form of inclusion body was about 25% of total proteins of E. coli. BL21(DE3). The inclusion body was washed once with 0.5% Triton X-100 and dissolved with 0.5% SKL, after renaturation by gradient dialysis, the recombinant protein was purified by DEAE-Sepharose FF cation column and then detected with 12% SDS-PAGE, it exists mainly in the eluted peak with 300 mmol/L NaCl and has high purity. By using enzyme-linked immunosorbent assay (ELISA), the recombinant protein was examined for reactivity with immunoglobulin M (IgM) antibodies in 6 sera from patients infected with Toxoplasma gondii ., it was reactive with all the 6 sera but not with sera from normal people, these results showed that the recombinant chimeric antigen has good antigenicity and specificity and could be used for detection of IgM against Toxoplasma gondii. The expressed chimeric protein could be used for epidemic investigation of Toxoplasma gondii, blood donor screening, especially for detection of pregnant women, and is of great significance in prevention of Toxoplasma gondii infection. |
The cytoplasm of E. coli is a reducing environment where cysteines do not engage in disulfide bonds. Any disulfide bonds that do appear are rapidly reduced through the action of disulfide reducing enzymes such as thioredoxin and glutaredoxin. To study the influence of E. coli cytoplasm on the solubility of recombinant proteins produced in it, bovine fibroblast growth factor (BbFGF), with single disulfide bond, and anti-HBsAg single-chain Fv (HBscFv), with two disulfide bonds, were selected as the pattern molecules of simple protein and complex protein, respectively. pJN98-BbFGF, a BbFGF expressing plasmid based on the vector pET3c, was constructed and transformed into normal host BL21(DE3) and a reductase deficient strain, E. coli Origami(DE3). At the same time, pQE-HBscFv, a HBscFv expressing plasmid was constructed and transformed into M15 [pREP4] and Origami(DE3). The recombinant BbFGF and HBscFv were produced in 2 types of bacteria and their solubilities and bioactivities were determined, respectively. It was found that the majority of BbFGF had formed inclusion body in the cytoplasm of BL21 (DE3) and all of them turned into soluble protein in Origami(DE3). It was also found the productivity of BbFGF in Origami (DE3) was 5% - 10% of the total protein and the value was 15% - 23% in BL21(DE3). BbFGFs produced in 2 recombinant bacteria were purified by cation exchange and heparin affinity chromatography. MTT assay revealed that the bioactivity of BbFGF purified from Origami(DE3) was higher than its counterpart from BL21(DE3). The ED50 of BbFGFs from different bacteria was 1.6ng/mL and 2.2ng/mL, respectively. As far as HBscFvs, both of them formed inclusion body in the cytoplasm of M15 [pQE-HBscFv] and Origami [pQE-HBscFv]. The inclusion body was solubilized in 6mol/L GuHCl, purified with a His-Trap column and then refolded by dialysis step-by-step against buffers containing downtrend concentration of GuHCl. Indirect ELISA was applied to determine the HBsAg binding activity of HBscFvs. It was found there was no obvious difference between the bioactivity of refolded HBscFvs produced from 2 recombinant bacteria. On the other hand, the supernatant of Origami [pQE-HBscFv] lysate displayed weak bioactivity and its counterpart from M15 [pQE-HBscFv] displayed without any bioactivity. The soluble HBsFv in the cytoplasm of Origami [pQE-HBscFv] was purified by cation exchange and immobilized metal affinity chromatography (IMAC) and the yield was 1 - 2mg/L. Those results suggested that modification of the redox environment of E. coli cytoplasm greatly improved the solubility of recombinant disulfide-bonded proteins produced in it. In the next step, we had like to co-express of molecular chaperones or refoldase to raise the yield of soluble recombinant proteins, as well as optimizing the culture condition of the "oxidizing" E. coli. |
Hepatitis E is an acute hepatitis casused by hepatitis E virus (HEV) in developing countries, where it occurs as cases sporadic and in epidemics form. The causative agent, hepatitis E virus, is transmitted primarily by the fecal-oral route. HEV is icosahedron non-enveloped virus, and its genome is a single-stranded, positive-sense, 3'-polyadenylated RNA about 7.5 kb in length. It contains three open reading frames (ORFs). Of which ORF1 codes for a polyprotein of 1693 amino acids and contain domains homologous to a viral methyltransferase, a papainlike cysteine protease, an RNA helicasre, and an RNA-dependent RNA polymerase, besides the most hypervariable region of the HEV genome. And ORF3 codes for a 123-amino-acide-long polypeptide with unknown function. While the major viral capsid protein (pORF2, ORF2 codes) of 660 amino acid was showed to contain the protective epitope. The bacterially expressed polypeptide disignated as NE2 has been proved to be a protective antige. And the anti-NE2 monoclonal antibodies (mAb) was screend, two of these mAbs 8C11 and 8H3 were showed to be against separate conformational neutralization epitope of hepatitis E virus (HEV). And these two mAb were used to screen for binding peptides from a 7-peptides phage display library. After four rounds of panning, tweenty-one positive monoclonal phages (11 for 8C11, and 10 for 8H3) were selected and the inserted fragments were sequenced. The DNA sequence coding for the obtained dominant peptide 8C11 (N'-His-Pro-Thr-Leu-Leu-Arg-Ile-C', named 8C11A) and 8H3 (N'-Ser-Ile-Leu-Pro-Tyr-Pro-Tyr-C', named 8H3A) were then synthesized and cloned to insert between amino acid 78 to 83 of hepatitis B core antigen (HBcAg), then expressed in E. coli. The recombinant proteins aggregate into homodimer or polymer on SDS-PAGE, and could bind with mAb 8C11 and 8H3 in Western blotting. Respectively, the recombinant protein C8C11A showed to be dimer mainly, which can bind with mAb 8C11. The monomer and dimer of C8H3A are in the same amount on SDS-PAGE, but only the dimer could bind with mAb 8H3 on Western blotting. The renatured recombinant proteins were all showed to aggregate into virus like particles which were similar as HBcAg on transmission electron micrograph. The dominant peptide 8H3A (N'-Ser-Ile-Leu-Pro-Tyr-ProTyr-C') that selected out by mAb 8H3 was further chemo-synthesized, and its binding activity was confirmed by BIAcore biosensor. The result showed that this 7-peptide can bind with mAb 8H3 in a big Ka and Kd form, which means the binding is not stable. These results implicated that conformational dependent neutralization epitope could be partially modeled by short peptide, which provided a feasible route for subunit vaccine development. |
Apolipoprotein A- I is the major apolipoprotein in high-density lipoprotein known to have a wide range of physiological functions, the best-studied one of which is in regulating cholesterol metabolism and preventing arteriosclerosis. Human blood has been the only source of this protein. To facilitate further research and application, it is essential to produce it through genetic engineering. In the current research, the baculovirus-insect cell system was used to overexpress human apolipoprotein A- I . Two recombinant baculoviruses were constructed. The first one expressed a pro form of apoA- I lacking native signal peptide. The recombinant protein was found to remain mainly inside cells in the early phase of infection, while being largely excreted to the medium late in infection. The second one used a heterologous signal peptide, snake phospholipase A2 inhibitor alpha subunit signal peptide, to lead the secretion of mature apoA- I. In contrast to the first virus, recombinant apoA- I was found in the culture medium at the early phase of virus infection. The mature apoA- I was purified from culture medium using Phenyl Sepharose hydrophobic interaction chromatography (HIC) and eluted with water and Propylene. This work shows that snake phospholipase A2 inhibitor a subunit signal peptide can be used to secret human apoA- I in insect cells, but the efficiency of its secretion is limited when the expression level is high. |
Glutamate dehydrogenase (GDH) is a key enzyme in the biosynthesis of glutamate. The GDHs from Corynebacterium glutamicum S9114 the most commonly used strain in glutamate fermentation, were purified and their molecular structures and properties characterized. The coenzymes were also studied in the hope to increase glutamate production. Cells were harvested at mid-exponential phase by centrifugation and washed with Tris-HCl buffer containing DTT and EDTA (pH 7.5). The cells were then disrupted using a French pressure cell press and the supernatant was collected by centrifugation. The extract was concentrated by 70-fold using the AKTA-100 FPLC system employing a DEAE-cellulose ion exchange column, a hydrophobic interaction chromatography (HIC) and Sephadex G-200 gel filtration. The purified extracts contained NADPH-dependent GDH and NADH-dependent GDH. Both of the enzymes were highly specific for the coenzymes. The molecular masses of the NADPH-dependent GDH and its subunit were 188kD and 32kD respectively, suggesting the enzyme is a homo-hexamer. Our data reported for the first time the presence of NADH- dependent GDH in Corynebacterium glutamicum S9114, similar to other microorganisms containing both GDHs. The NADPH-dependent and NADH-dependent GDH in Corynebacterium glutamicum S9114 may participate in the assimilation and dissimilation of ammonia respectively. The absorptions of NADPH-dependent GDH was very weak at 280nm but very high at 215nm, suggesting a low phenylalanine and tyrosine content in the enzyme. |
Human tumor necrosis factor alpha (hTNF-alpha) is one of the most important inflammatory cytokines that acts as a mediator in inflammatory and immune response and plays a key role in host defense against infection. The over expression of hTNF-alpha is associated with serious consequences, such as shock, hypotension, thrombus, septicemia and even death. It has been implicated in many autoimmune and inflammatory diseases, such as rheumatoid arthritis, Crohn's disease, chronic heart failure and septic shock. Inhibiting the bio-activity of hTNF-alpha is one of the strategy for the treatment of these diseases. Compared with traditional recombinant protein drugs, small molecule drugs have many advantages, such as high affinity, low immunogenecity and low cost. Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful method for the selection of oligonucleotides that bind with high affinity and specificity to target proteins. Such oligonucleotides are called aptamers, and are potential therapeutics for blocking the activity of pathologically relevant proteins. To obtain oligonucleotide aptamers specifically binding to TNF, a 40nt random DNA combinatorial library flanked by 31nt fixed sequences was chemically synthesized. The random library was amplified with PCR and subjected to selection by SELEX protocol against hTNFalpha. After incubation of the library with hTNFalpha, the mixture was blotted onto Immobilon-NC transfer membrane. The no-specific binding was washed away and the hTNFa binding aptamers were eluted and detached from the target protein. The eluted oligo nucleotides were amplified with PCR and served as the DNA library for the next round selection. After 12 rounds of such selection, the selected aptamers were cloned to pGEM-T vector. Positive clones were identified by restriction enzyme digestion and DNA sequencing. Oligo DNA were synthesized according to the sequence data and tested for their activities. Binding activity of the aptamers to hTNFalpha were detected by ELISA and dot blot with biotin-streptavidin-horseradish peroxidase system. Mouse L929 cells were used to test the anti-hTNFa activity of the DNA aptamers. The aptamers were incubated with hTNFalpha and added to the L929 cells. The results were read under microscope and with MTT staining. It was shown that these DNA aptamers bound to hTNFalpha with high affinity, and can inhibit the cytotoxicity of hTNFalpha on cell culture. The affinity of these aptamers are different and may related to their structure. These ssDNA aptamers are potential for the treatment and diagnosis of hTNFalpha related diseases. |
A large amount of alpha-ketoglutarate (alpha-KG) (6.8 g/L) was accumulated in flask culture when CaCO3 was used as a buffering agent in the production of pyruvate by multi-vitamin auxotrophic yeast Torulopsis glabrata CCTCC M202019. In a 5 L jar-fermentor, less alpha-KG (1.3 g/L) was produced when NaOH was used to adjust the pH, while more alpha-KG (11.5 g/L) detected when CaCO3 was used as the buffer. In the latter case, the molar carbon ratio of pyruvate to alpha-KG (C(PYR)/ CalphaKG) was similar to that obtained in flask culture, suggesting the accumulation of alpha-ketoglutarate was related to the addition of CaCO3. Furthermore, it was found that: (1) delaying the addition time of CaCO3 decreased the a-ketoglutarate formation but increased C(PYR)/ C(alphaKG); and (2) under vitamin limitation conditions increasing the concentration of CaCO3 led to an increased a-KG accumulation at the expenses of pyruvate. To study which ions in CaCO3 was responsible for the accumulation of alpha-KG, the effects of different pH buffers on the a-KG accumulation were studied. The level of alpha-KG was found to correlate with the levels of both Ca2+ and CO3(2-), with Ca2+ played a dominant role and CO3(2-) played a minor role. To find out which pathway was responsible for the accumulation of alpha-KG, the effects of biotin and thiamine on alpha-KG accumulation was investigated. The increase in biotin concentration led to an increase in alpha-KG accumulation and a decrease in C(PYR)/ C(alpha-KG), while the levels of alpha-KG and C(PYR)/C(alphaKG) were not affected by thiamine concentration. The activity of pyruvate carboxylase was increased as much as 40% when the medium was supplemented with Ca2+ . On the other hand, the activity of the pyruvate dehydrogenase complex was unaffected by the presence of Ca2+. To conclude, the higher level of a-KG was caused by higher activity of pyruvate carboxylase stimulated by Ca2+, with CO3(2-) served as the substrate of the reaction. |
Two strategies, direct ligation after enzyme digestion and over-lap PCR technology, were adopted to construct a fusion gene which was composed of the antimelanoma single chain antibody gene and the staphylococcal enterotoxin A gene without N-terminal signal sequence. The fusion gene was subcloned into pET28-a vector and transformed into E. coli BL21(DE3). Ni-NTA system was selected to separate and purify the expresstd products. The inhibition ratio of the fusion protein was tested by MTT method. It is shown that the 6His-ScFv-SEA fusion protein can be expressed stably in E. coli BL21 (DE3). The quantity of the fusion protein was shown up to 30% of the total protein of the bacteria and mainly in inclusion body. By activation the effective cells, the fution protein can inhibit the melanoma cell whith expressed corresponding antigen. |
Human Zona Pellucida(ZP), which is a complex matrix surrounding oocytes,is comprised of three immunologically distinct glycoproteins(hZP1, hZP2 and hZP3). Because hZP3 possesses the sperm receptor activity and the acrosome-inducing activity, it has long been used as a candidate antigen to develop an immunocontraceptive vaccine. However, a large amount of native hZP3 protein is unavailable. It is an effective way to express hZP3 protein directly in vitro. Nevertheless, it had been reported that the rhZP3 protein produced in Pichia pastoris was not secreted but accumulated in the cells and could only be purified after being solubilized by strong denaturants. More unfortunately, after purification the final product required 6mol/L urea to maintain solubility. An improved project was advanced with the aim to express secreted and soluble rhZP3 protein in yeast. In this study, the fragment of hZP3 cDNA coding for aa 23 - 408, which the N-terminal leader was removed and most of the C-terminal transmembrane-like domain was reserved, was amplified by two PCR primers including EcoR I and Not I sites respectively and a His6 codon cassette was added to 5'-terminal. The hZP3 insert was incorporated into expression vector pPIC9K. The resulting recombinant yeast expression vector was designated pPIC9K-rhZP3. Linearized pPIC9K-rhZP3 was transformed into Pichia pastoris. After G418 selection, the recombinant Pichia pastoris strains were identified by PCR and the rhZP3 was expressed following the manufacturer' s protocol. Following induction with methanol, the rhZP3 protein was secreted and dissolved into the culture supernatant. SDS-PAGE and Western blot analyses showed that the apparent molecular weight of the expressed rhPZ3 proteins in yeast was smaller and a little size heterogeneity than native ones; after purified with Ni-chelating affinity chromatography, the final product's apparent molecular weight was about 32 - 34KD and their yield more than 20mg/L. We supposed that the C-terminal transmembrane-like domain be useful for secretion of rhZP3 into the culture supernatant and the expressed rhZP3 protein be incompletely digested by proteinases of Pichia into shorter fragments which all were glycosylated inhomogeneously. Fortunately, the fragments of rhZP3 protein can be recognized in Western blot by the polyclonal antibodies to porcine ZP3 which has showed a cross-reactivity with human ZP in vitro. It will be expected that the rhZP3 protein expressed in Pichia pastoris not only has immunogencity, say, it can rise antibodies in vivo to prevent spermatozoa-ovum binding, but also does not contain ovarian factors that might be the cause of undesired side effects, e.g. ovaritis and can be used as a safe immunogen in human antifertility vaccine research. |
An expression plasmid carrying anthrax protective antigen (PA) gene was constructed, which has an OmpA signal sequence attached to the 5' end of PA gene. The plasmid was transformed into E. coli and induced to express recombinant PA (rPA) . The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ion-exchange, hydrophobic interaction chromatography, and gel filtration, about 15 mg of 95 % pure rPA was obtained from 1-liter culture. The bioactivity of rPA was proved by in vitro cytotoxicity assay. The polyclonal antiserum from rabbits immunized with rPA could inhibit the action of anthrax lethal toxin in vitro, which suggests that antibodies against rPA can provide high passive protection against anthrax. The results reported here may be helpful to develop a safe and efficacious recombinant PA vaccine against anthrax. |
Conjugal plasmid pGH112 has been developed based on the replicons of Streptomyces coelicolor plasmid SCP2 and E. coli ColE. The plasmid contains ampicilin resistance gene(amp) for selection in E. coli and thiostrepton resistance gene (tsr) for selection in Streptomycetes, and a 0.76 kb oriT fragment of (IncP) RK2. Conjugal transfer of pGH112 was performed from E. coli to S. coelicolor A3(2), S. avermitilis, S. lividans TK54, S. toxytricini NNRL15443, S. venezuelae ISP5230 and Sacc. erythraea by conjugation, results show that the plasmid was able to transfer efficenctly from E. coli to Streptomycetes, was stably inherited in the recipients. pGH113 was constructed from pGH112 by combining the constitutive ermE promoter with green fluorescent protein gene(gfp). |
The CrylA Crystal Protein from Bacillus thuringiensis is associated with DNA, but the role and sequences of these DNA molecules are unknown. CrylA bipyramidal crystals from B. thuringiensis strain 4.0718 was selectively dissolved and associated DNA was extracted from protoxin. The DNA was digested with Nde I to obtain 3 to 5 kb fragments and then the fragments were subcloned into pMD18-T vector, screening of recombinants were done by PCR-RFLP and sequencing. The ORF of cry1Ac gene was amplified by primers designed and then subcloned. The 3.5 kb BamH I and Sal I fragments of pMDX35 was inserted into the pET30a vector, giving 8.9 kb recombinant plasmid, pETX35. ETX35 strain were obtained by transformed pETX35 into B121 (DE3). A 141 kD fusion protein was superexpressed as inclusion bodies. Quantitative protein analysis indicated that the amount of 141 kD protein was above the level of 51.36% of total cellular protein. Plasmid pHTX42 constructed from shuttle vector pHT304 was transformed B. thuringiensis acrystalliferous strain XBU001 with electroporation to obtain the recombinant HTX42. The recombinant protein was found with a molecular mass of 130 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Scanning analysis indicated that the expressed protein accounted up to 79.28% of total cellular proteins and accumulated in the cells mounted up to 64.13% of cellular dry weight. Under Atomic Force Microscopy (AFM), typical bipyramidal crystals from HTX42 strain were found with a size of 1.2 microm x 2.0 microm. Bioassay showed that these inclusion bodies of ETX35 strain and crystals from HTX42 strain were highly toxic against the larvae of Plutella xylostella. On such a base, constructing insecticidal recombinant and analyzing the source, structure, and function of the 20 kb DNA can be further achieved. |
The use of tumor antigen specific antibody for the delivery of therapeutic agents offers the possibility of targeting therapy with reduced toxicity to normal tissues compared to conventional treatments. In previous work, the human-mouse chimeric antibody fragment Fab' directed against CD20 was constructed from the new anti-CD20 antibody HI47 (a mouse IgG3, K). The chimeric antibody fragment Fab' could reduce its antigenicity, but the yield, quality and affinity of chimeric antibody fragment Fab' restrict its use. To improve affinity of chimeric antibody fragment Fab', a new phasmid pYZcpp3, which expresses chimeric antibody fragment F(ab')2, was constructed by adding a sequence encoding a small peptide, (CPP)3, to C-terminus of heavy chain constant region of chimeric antibody fragment Fab'. Using the pYZcpp3 to transform E. coli. 16c9, the genetically engineered bacteria 10916# was obtained. 10916# can secret the soluble chimeric antibody fragment Fab' and F(ab')2 into periplasmic. The yield was up to 360 mg/L with the percent of F(ab')2 up to 45% in 19L fermentor by the high density fermentation technology. Without denaturation and renaturation, the F(ab')2 has possessed the native three-dimensional structure. The purity of F(ab')2 was more than 90% after the purification of protein G affinity chromatography and S200 size exclusion chromatography. The F(ab')2 could distinguish and bind to Raji cells (CD20+) by FACS. F(ab')2 could inhibit the proliferation of Raji cells in vitro by MTT, IC50 was 22.8 microg/mL. HI47 and its chimeric fragments F(ab')2 induced a significant level of apoptosis (23.5%, 20.8%, respectively), independent of any cross-linking agents, in Raji cells after 24 h incubation. The chimeric antibody fragment F(ab')2 directed against CD20 is possible to apply to tumor therapy in clinic in the future. |
The development of the systematic evolution of ligands by exponential enrichment (SELEX) process has made it possible to isolate oligonucleotide sequences with the capacity of recognizing virtually any class of target molecules with high affinity and specificity. These oligonucleotide sequences, referred to as "aptamers", are useful as a class of molecules that rival antibodies in diagnostic applications. Aptamers are different from antibodies, yet they mimic properties of antibodies in a variety of diagnostic formats. To meet the shortcomings of antibodies, aptamers have the following advantages. Aptamer does not depend on animals, cells, or even in vivo conditions and produced by chemical synthesis with extreme accuracy and reproducibility. Once denatured, functional aptamers could be regenerated easily within minutes. They are stable to long-term storage and can be transported at ambient temperature. We describe here an enzyme -linked oligonucleotide assay that use a SELEX-derived RNA aptamer to detect hTNFalpha. In order to protect from nuclease attack, the RNA aptamer was modified by replacement of 2'-NH2 for 2'-OH at all ribo-purines. In a sandwich micro-plate assay, hTNFalpha monoclonal antibody was coated on the surface of the plate, biotin-labeled RNA aptamer was used as a detect molecle. HTNFalpha was diluted by pooled human serum as standard, and streptavidin-horseradish peroxidase-substrate system was added for detection. Accuracy, precision, sensitivity, specificity of ELONA method were analyzed. The levels of hTNF-alpha in normal human serum samples were assayed by the ELONA and the ELISA processes. The resultes demonstrate that a sandwich assay using a SELEX-derived RNA aptamer has parameters for accuracy, precision, sensitivity, specificity well within the limits expected of a typical enzyme-linked assay. There is no significant difference between the results of ELONA and ELISA. The minimum detection level was 100 pg/mL. This method will be useful for detection of almost all the cytokines and other protein molecules. |
In order to study the feasibility of E2 gene fragment of hepatitis virus G(HGV) as a component of DNA vaccine against the hepatitis virus G infection, a 559bp DNA fragment encoding HGV E2 was cloned into plasmid pCMV-S from pThioHis-E2 in the same reading frame with HBsAg gene to form a recombinant plasmid named pCMV-S-E2. BALB/c mice of Kunming strain were immunized with purified plasmid DNA of pCMV-S-E2 by intra-muscularly inoculation. The immunizations were boosted twice at an interval of 14 days. The whole blood was collected from mice orbit on the day-8 after the last boost. Mice sera were screened by ELISA to determine the humoral immune response using E2-GST fusion protein as the immobilized antigen and the sera from mice immunized with pCMV-S as control. The result indicated that the immunization with plasmid DNA of pCMV-S-E2 could induce quite strong humoral immune response. |
Diphtheria toxin A fragment (DTA) is an essential catalytic domain of diphtheria toxin (DT)-based immunotoxin. DTA protein and its antibodies play an important role in the studies on toxicology, purification and identification of DT-based immunotoxins. In this paper, DTA was expressed and purified from E. coli. After Q-Sepharose FF chromatography and (Ni+)-Sepharose affinity chromatography, 6 x His-DTA fusion protein with 90% purity was achieved. Using the purified DTA as antigen to immunize BalB/c mice, 2 hybridoma cell lines (designated as 3B6 and 3B9, respectively) secreting monoclonal antibodies (McAbs) against DTA were established. Investigations showed that both McAbs were characterized as IgG1 with titers of 1: 10(6). The binding of the McAbs to DTA was competitively inhibited by horse sera against DT. The fact that anti-DTA McAbs could be used in western blot analysis and affinity chromatography purification of DT-based immunotoxins implied that they will be useful agents in the studies on DT-based immunotoxins. |
Prostate stem cell antigen (PSCA), a homologue of the Ly-6/Thy-1 family of cell surface antigen, is expressed by a majority of human prostate cancers and is a promising target for prostate cancer immunotherapy. To obtain the specific peptide binding with PSCA for targeted immunotherapy, PSCA gene was obtained by RT-PCR from human prostate cancer cell line DU145 and the transcated PSCA (tPSCA) gene was cloned into vector pQE30 for soluble expression in E. coli. The identity of recombinant tPSCA was confirmed through ELISA and western blot by use of anti-PSCA monoclonal antibody. Then the 12-peptide phage display library was screened with the purified tPSCA protein for its specific binding peptide through 3 rounds panning. For identifying the peptide's specificity, the peptide was coupled with EGFP (enhanced green fluorecent protein) by recombinant DNA technology and the recombinant coupled protein was termed 11-EGFP. The binding specificity with tPSCA of 11-EGFP was further confirmed by ELISA and competitive inhibition experiment. Flow cytometry demonstrated its binding specificity with cell line DU145. In conclusion, a 12-amino-acid peptide which could bind with PSCA specifically was found and it may be a potential tool for targeted immunotherapy of prostate carcinoma. |
The negative signal provided by interactions of costimulatory molecules, programmed death-1 (PD-1) and its ligands, PD-L1 (also B7-H1) and PD-L2 (also B7-DC), is involved in the mechanisms of tumor immune evasion. To block PD-Ls-PD-1 interactions by a soluble receptor of PD-1, we constructed a eukaryotic expression plasmid that expresses extracellular region (aa1-aa167) of murine PD-1 (pPD-1A) and, another version of pPD-1A, pPD-1B, carrying cDNAs encoding for both extracellular region of PD-1 and green fluorescent protein (GFP) reporter gene, which was inserted downstream of PD-1. Experiment of BHK cells transfected with pPD-1B determined that most expression product (sPD-1) in the cells was secreted out. FACS analysis revealed that sPD-1 was specific and bound efficiently to PD-1 ligands. Cytotoxicity assay showed that blocking PD-Ls on either tumor cells or spleen cells by sPD-1 mediated enhanced lysis of H22 cells by Hsp70-H22 peptides complexstimulated spleen cells. The constructed plasmid vector would provide a novel method of tumor gene therapy of blocking PD-Ls-PD-1 interactions by expression of soluble receptor of PD-1 in tumor sites, which could increase the antitumor activity. |
It was constructed that a genomic DNA library from Lactobacillus sp. MD-1 yielding D, L-lactic acid. The gene encoding L-lactate dehydrogenase (L-LDH) was cloned from the genomic library of strain MD-1 by complementation in E. coli FMJ144 which was lactate dehydrogenase and pyruvate-formate lyase double defective mutant. The nucleotide sequence of the ldhL gene predicted a protein of 316 amino acid starting with ATG. The putative molecular weight of the L-LDH amino acid sequence was 33.84kD. A putative typical promoter (-35 and -10 boxes) had been observed in the 5' noncoding region. An rho-independent transcriptional terminator has been observed in the 3' noncoding region. Three highly conserved regions (Gly13 approximately Asp50, Asp73 approximately Ileul00 and Asn123 approximately Arg154) with several conserved residues had been identified. Gly13 approximately Asp50 was NADH-binding site domain. Asp73 approximately Ileu100 and Asn123 approximately Arg154 were reported to be the active site domains. The ldhL and the L-LDH of Lactobacillus sp. MD-1 showed the low identity and similarity with other Lactobacilli, and the highest percentage were 61.9% and 68.9% respectively. All the above indicated this gene is a novel ldhL. |
The expression of the vgb gene in vivo could improve the fermentation density and then contribute the extracellular secretion of the product of bxn gene. Constructed the recombination plasmid pPIC9K-vgbbxn and transformed into Pichia pastoris GS115. The results of PCR and SDS-PAGE indicate that the vgb gene and bxn gene had integrated into the genome of Pichia pastoris GS115 and expressed in efficient level. Also, the protein activity of their products had been verified respectively. Shake flask fermentation experiments showed that the presence of VHb in yeast Pichia pastoris efficiently enhanced cell growth and secretive expression of bxn gene under hypoxic habitats. |
Considering Alcaligenes faecalis pencillin G acylase(AfPGA), which possesses the attractive characteristics for beta-lactam antibiotics conversions, the gene of PGA was cloned into an expressing vector pKKFPGA. The recombinant plasmid contained multicopy replicon(COLE 1), trc promoter, AfPGA gene, rrnB transcript terminator and ampicillin marker transformed Escherichia coli DH5alpha. As both the recombinant plasmid and the host DH5alpha had no laclq gene, the trc promoter was always active and the AfPGA could be constitutively expressed without IPTG induction in the host DH5alpha. In the shaking flask, the recombinant cell was inoculated into the fermentation medium (tryptone 10g/L, yeast extract 5g/L, MgSO4 x 7 H2O 1g, KH2 PO4 2g/L, K2HPO4 x 3H2O 5g/L, Na2HPO4 x 12H2O 7g/L, (NH4)2SO4 1.2g/L, NH4Cl 0.2 g/L, NaCl 0.1g/L, dextrin 30g/L) and cultured at 28 degrees C for 20h. The production of AfPGA reached 2,590u/L(NIPAB method), with a cell-density-specific activity of more than 300(u/L)/A600, this yield increased 432 fold higher than the native expression of Alcaligenes faecalis . Without ammonium sulphate fractionation and dialysis, the supernatant of crude extract was directly loaded on DEAE-Sepharose CL 6B column equilibrated by phosphate buffer (50mmol/L, pH7.8), and the enzyme fraction was not absorbed on the column but impurities were absorbed. Subsequently the effluent was added ammonium sulphate to 1mol/L and loaded on Butyl-Sepharose CL 4B column equilibrated by 50mmol/L phosphate buffer pH7.8-1mol/L ammonium sulphate. The enzyme was eluted as concentration of ammonium sulphate in phosphate buffer decreased to 0, PGA was eluted. After these two column chromatography, the enzyme was enriched 20 times with a 91% activity recovery. The purified enzyme had a specific activity of 68.6u/mg protein. However, the overproduction of PGA was often limited by translocation and/or periplasmic processing steps, subsequently resulted in intracellular accumulation of various types of PGA precursors and then formed inclusion bodies in the cytoplasm and/or periplasm. In this study, 5% PGA precursors formed as inclusion bodies in the cytoplasm while no inclusion bodies formed in the periplasm. It suggested most PGA precursors were transported to the periplasm and matured to active PGA and also explained why PGA gene was highly expressed in the host DH5alpha. On the other hand, inclusion bodies in the cytoplasm indicated that the maturation of PGA in the host DHSalpha was limited by the translocation step. |
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