The single-chain Fv cDNA of a monoclonal antibody specific for glycoprotein of rabies virus, was cloned into the PE40 (a truncated form of pseudomonas exotoxin) gene inserted fusion protein expression plasmid pET-PE40 to form the expression vector pET-ScFv-PE40, The recombinant plasmid was identified by restriction enzyme digestion and sequencing.After transfection of E.coli BL21 and induction by IPTG,the recombinant immunotoxin fusion protein was highly expressed in E.coli BL-21 in the form of inclusion bodies(up to 32.29% of total cellular proteins). The fusion protein was refolded by constant gradient dialysis and purified with anion exchange chromatography, hydrophobic interaction and gel filtration chromatography, the interest protein was about 96% pure. Indirect immunofluorescent staining showed that recombinant immunotoxin could specifically bind to rabies virus-infected cells. Cytotoxicity analysis by MTT showed that the fusion proteins could kill rabies virus-infected cells specifically,while having no toxicity to normal cells.
The matrix protein (M) of human respiratory syncytial virus (RSV) is important for virion morphogenesis and contains CTL epitopes valuable to the research on RSV vaccine development. Therefore, M gene was amplified from RSV infected HEp-2 cells by RT-PCR, and FGAd/RSVM of the replication deficient recombinant adenovirus containing M gene was constructed and identified. DNA sequencing displayed no nonsense mutation in M gene. From the FGAd/RSVM infected 293 cells, the transcription and expression of M gene were confirmed by RT-PCR, indirect immunofluorescence assay and Western blotting, respectively. The resulting FGAd/RSVM laid a solid foundation for the investigation into the protective immunity against RSV infection and its mechanism.
This study was aimed to investigate the mechanism of indoleamine 2,3-dioxygenase(IDO) in HepG2 cells contributing to tumor immune escape. Methods T-lymphocyte and HepG2 cell were cocultured, and 1-methyl-tryptophan(1-MT) was added to make an intervening experiment. The expression of IDO mRNA in HepG2 cell cocultured with T-lymphocyte was detected by RT-PCR ;the apoptosis rate of HepG2 cell cocultured with T-lymphocyte was analyzed by flow cytometer; The cytotoxicity of T-lymphocyte against HepG2 cell was examined by MTT assay. Results In combine reaction system , the expression of IDO mRNA was strongly induced in HepG2 cell cocultured with T-lymphocyte and faintly induced when cultured with 1-MT ; the earlier apoptosis rate of HepG2 cell in control group and experiment group(1-MT:1.25 mmol/l，2.5 mmol/l，5mmol/l) was respectively（3.48±0.34）%，（7.82±0.41）%，（18.62±0.42）%，（25.32±0.40）%（P<0.01），The cytotoxicity of T-lymphocyte against HepG2 cell was respectively（17.36±1.24）%，（25.48±1.48）%，（32.89±1.73）%，（42.04±2.16）%（P<0.01）. Conclusion These results suggest that IDO from HepG2 cell can suppress the tumor immunity of T-lymphocyte to kill hepatoma carcinoma cell, which may be contributing to hepatoma immune escape
Object： To improve the genetic stability of HBV gene in transgenic mice．Methods：HBV transgenic mice were bred by backcross and double cross . The HBV gene expression and replication were studied with real-time PCR,ELISA and chemiluminescence．Results:The HBV transgenic mice have stably bred to 23rd generation．The serum HBsAg level is 4122.31±2044.74IU/ml; The rate of HBV transgenic mice whose serum HBV DNA reach 104-106copies/ml was 93.93%.The HBV replication and expression were improved markedly.There is no difference between male and female mice about serum HBsAg level. Conclusions:After breeding the HBV gene was expressed stably with high-level in transgenic mice.
To investigate the influence of initial copolymer compositions of PLGA on mechanical property, degradation behavior and biological properties of the scaffolds. Porous PLGA scaffolds with different initial copolymer compositions were prepared by solvent casting/particulate leaching method. Biomechanical properties were measured using testing machine and degradation rate was detected by soaking the scaffolds in phosphate buffered solution at 37℃ for various time. Human dermal fibroblasts were seeded on PLGA scaffolds with different initial copolymer compositions and incubated for various time points. Cell adhesion efficacy, proliferation rate, and total collagen content were analyzed and cell morphology on the scaffolds was observed using scanning electron microscopy. The results showed that with the increase of PLA ratio, mechanical strength of the scaffolds increased, whilst the degradation rate decreased, but the changes were not lineal. PLGA 70:30 scaffolds got the highest mechanical strength, and there were no significant difference on the degradation rate between PLGA 70:30 and 80:20. PLGA scaffolds with different initial copolymer compositions had good cytocompatibility, showing high cell adhesion efficacy, fast proliferation rate and stretched cell morphology with no significant difference. A large amount of extracellular matrix was secreted and after 7 days of culture, cell nearly covered all the surface of the scaffolds. Based on these results, the study drew the conclusion that the copolymer compositions of PLGA affected the mechanical, degradation and biological properties of the scaffolds subtly but significantly. This would be helpful for choosing PLGA scaffold materials for constructing tissue.
Plants are constantly challenged by various biotic and abiotic stresses, such as disease, drought, high salinity, and low temperature in their natural environment, which cause adverse effects on the growth of plants and serious reductions in the quantity and quality of crops. AR2/ERF is a large family of transcription factors. Genes in the ERF family encode transcriptional regulators with a variety of functions involved in the developmental and physiological processes in plants. Three AP2/ERF family transcriptional regulators (BnaERF1, BnaERF2 and BnaERF3) were isolated from Brassica napus by in silico cloning method using the conserved domain amino acid sequence of Arabidopsis thaliana ERF as probe. Then, cDNA and deduced amino acid sequence, composition, hydrophobicty and hydrophilicity, physical and chemical characterization, phylogenetic tree, conserved domain sequences, function domain, molecular modeling, and folding state were predicted and analyzed. Then, we isolated BnaERF1, BnaERF2 and BnaERF3 genes from B. napus L. Huyou15 by RT-PCR and PCR using cDNA and DNA as template.
15-hydroxyproglandin dehydrogenase(PGDH) is a tumor suppressor gene. PGDH transcript and protein are both highly expressed by normal tissue but are nearly undetectable in several tumors. Total RNA was isolated from human normal colonic epithelia. Then the PGDH cDNA gene was amplified by reverse transcript polymerase chain reaction (RT-PCR). The amplified fragment was orientationly linked into the prokaryotic expression vector pBV220 by T4 DNA ligase and then sequenced. The result indicated that the cloned gene was a corrected PGDH. Then the recombined expression plasmid was transduced into DH5a by TSS, and the expression of PGDH protein was induced by temperature for 3 hours at 42 C, and the fusion protein of PGDH-His6 was analyzed by SDS-PAGE and Western blot after lysis of bacteria. The SDS-PAGE result showed that cloned recombinant protein was expressed in the inclusion body with molecular weight of approximated 29kDa. The PGDH fusion protein containing about 30% of total somatic protein, was stably expressed in E.coli. After ultrasonication and purification by immobilized metal chelation affinity chromatography, homogenous protein was obtained and reached 95% purity as proved by SDS-PAGE. Western blot identified its specificity. The expressed PGDH protein had highly bioactivity, and its enzyme activity is about 3.7×104U/mg.
DhaBCE from Klebsiella.pneumoniae was inserted into the yqhD upriver in the plasmid pET28(a+)-yqhD. The recombinant plasmid pET28(a+)dhaBCE-yqhD harboring the genes encoding dhaBCE and yqhD with a Shine-Dalgarno (SD) sequence was transformed into E. coli novablue to be co-expression. The results showed that after 16 hours calculated following the addition of inducer IPTG at induce temperature 28℃, the enzyme activity of glycerol dehydratase and yqhD. oxidoreductase in recombined strain could arrive 35 U/ mg and 82 U/ mg, individually, higher than the wild type. The yield of 1, 3-PD arrived at 39g/L when 55g/L of substrate glycerol was added in the fermentation culture. Excessive glycerol badly influence produce formation and inhibition of product on 1, 3-propenediol biotransformation existed.
This paper reported the role of Phosphoenolpyruvate Carboxylase of procaryote to the genetic regulation of lipids synthesis. A gene fragment (pepcA) containing the conservative sequence (Fa) of PEPC gene from filamentous cyanobacterium Anabaena sp. PCC 7120 was cloned and inserted between BamHI sites of the shuttle expression vector pRL-489 in forward or reversely orientation. The recombinants (pRL-Sen pepcA and pRL-Anti pepcA) were transferred into the host cells Escherichia coli DH5a. The results showed that the introduction of pepcA fragment hadn't obvious influence to the growth of transgenic E.coli. Compared to the wide cells, the enzyme activity of the transgenic E.coli of pRL-Anti pepcA reduced to 30.2%, the protein content of that was 23.6% lower than in wide cells, and the lipid content of that increased 46.9%. While the enzyme activity of the transgenic E.coli of pRL-Sen pepcA was 2.38 fold of wide cells, the protein content of that increased 14.5%，and the lipid content was lower 49.6% than in wide type cells; The content of C18 in transgenic E.coli increased obviously. The above results may offer a clue to produce biodiesel with procaryote.
To enhance the yield and productivity of cutinase from recombinant Bacillus subtilis WSHB06-07, the effect of pH on cell growth and product formation was investigated. Based on specific cell growth rate and specific cutinase formation rate under different pH condition, a two stage pH control strategy was developed, in which pH was controlled at 7.5 for the first 4h and then shift to 6.5. By the utilization of this strategy, the yield of cutinase was significantly improved, the maximal cutinase activity and the productivity were 170 U/mL and 16.9 kU/(L·h), which were increased by 122.6% and 123.2%, respectively, compared to that from the condition of constant pH 7.5.
The influence of dissolved oxygen (DO) concentration, varied from10% (of air saturation) to 60%, on glycerol production by Candida glycerinogenes in the batch fermentation was investigated by using the chemically defined medium. The results showed that it was suitable for glycerol production by C. glycerinogenes at DO concentration of 30%, the glycerol concentration, yield and productivity were 120.7 g/L, 0.575 g/g and 1.69 g/(L•h), respectively, while the glycolytic by-products formation was the least. At DO concentration of 10%, the “Pasteur effect” arose during the whole fermentation process, so the glycolytic by-products could be kept a high level. During the rapid growth phase, the cells of C. glycerinogenes could gradually transit their anaerobic fermentation to aerobic metabolism and reduce the formation of glycolytic by-products when the DO concentration increased from 10 % to 60 %. However, the oxygen consumption of cells decreased sharply during the steady growth stage. With increasing DO concentration, the amounts of ethanol and acetic acid were gradually decreased. Logistic equation, Luedeking-Piret equation and Luedeking-Piret-Like equation were successfully applied to estimate the kinetics of cell growth, glycerol production and glucose consumption, respectively, under diverse DO concentrations.
High abundant plasma proteins deterior the effect of seperating plasma proteins in plasma proteomics research, it is necessary to seperate high abundant proteins in plasma samples. Protein G were used to absorbe the anti-albumn monoclonal antibodies then cross-linked with mAb by Dimethyl pimelinediimidate chloride. The resulst suggested that 1000µg antibody were site-directed immoblized and kept the capacity of binding around 600µg albumin proteins after 10 cycles of elution-regeneration-binding.. The bound proteins were identified with 2-DE-MALDI-TOF/TOF. Bound proteins included albumin, IgG, Vitamin D binding protien, Fibrinogen, Transthyretin, Keratin 10, Transferrin, C3, Proapolipoprotein, CD5 antigen-like, Alpha1-Antitrypsin. Our study showed a prevailing,applicable and valuable approach to separate high abundant proteins in human plasma as well as other samples.
Hypermethylation of CpG islands is an epigenetic modification and plays key role in tumorigenesis . Several methods for methyl-CpG islands detection have been established , but high through methyl-CpG islands enrich method for whole genome was not reported till now. In this study we reported a rapid and efficient genome-wide methylation enrichment method which is based on DNA immunoprecipitation for accumulating methyl-CpG islands .The GST-MBD2b recombinant protein was expressed in E.Coli and purified by using Glutatione Sepharose 4B. The methylated CpG islands was enriched by the GST-MBD2b affinity column which has the capability to bind methylated CpG islands at high salt concentration. The enrich efficiency was detected by real time PCR and the results indicated that the methylated CpG islands was enriched more than 100 folds. The enriched methylated CpG islands could be used further experiments for example real time quantitative PCR, DNA sequencing and whole genome CpG islands microarray analysis.
A simple and effective method for the duplex PCR detection was developed by using sequences of exogenous gene (RDV MP-) and endogenous gene (Zein) as templates for PCR amplification. The results of routine PCR amplification for RDV MP- gene in transgenic maize suggested that RDV MP- gene can stably inheritate in transgenic plants and their progenies; The duplex PCR detection of all negative and part positive samples that obtained by routine PCR amplification confirmed that above negative results were exact, also showed that the quality of extracted DNA can meet the need of PCR amplification. The error ratio of negative samples was 1.4%. The method used in this study was simple and credible and can be used to detect transgenic plants and their products.
To construct a prokaryotic expression vector of Human SUMO-3, purify Human SUMO-3 proteins produced by the expression system, and prepare its antiserum. The fragment of human SUMO-3 gene was amplified from pEYPF-SUMO-3 plasmid by PCR. The enzyme digested target fragment was cloned into pET41a(+) clone and expression vector and transfected into E. coli. BL 21 (DE3) plysS, in which SUMO-3 expression was induced by IPTG. After the soluble protein was purified through GST affinity chromatography, processed by identified by SDS-PAGE, a rabbit was immunized with the fusion protein, and the antiserum was obtained. The result of DNA sequence analysis showed that the cloned SUMO-3 gene sequence was completely corresponding to GenBank data. SDS-PAGE and Western blotting showed that the expressed SUMO-3 fusion protein was about 44.0 kDa, mainly existing soluble protein of E. coli. , that could be purified through GST affinity chromatography. The results of ELISA is positive, and Western blotting confirmed that the antiserum reacted specifically to the SUMO-3 protein. A recombinant SUMO-3 protein and the specific polyclonal antibody have been obtained, which provides a basis for establishment of immunoassays of human SUMO-3.
After comparisons of the sequences of amino acids of HN gene among NDV strains with genotype difference, it was revealed that in strain Ⅶ the sequence of amino acid from 65 to 75 was conservative, while the same fragments in other strains were different from each other. So the fragments of HN gene in strain La Sota, GX-2 and F48E9, which were the representative strains of Ⅱ、Ⅶ、Ⅸ genotype of NDV, were cloned and expressed, while the antigenicities of expressed peptides were assayed by anti-serum. SPF chickens were immunized by each expressed proteins and the antibody titers of different groups were assayed by ELISA. The results showed that there were differences in reactionogenicity in 3 strains. All chickens which were immunized by different expressed proteins were challenged by F48E9, a velogen strain of NDV. Results showed that the protection rate of every expressed protein was different from each other, which illustrated that there were differences of immunogenicity in researched region among 3 strains.
Myostatin is a negative regulator of myogenesis, which inhibits myogenic cell differentiation by down-regulating MyoD expression, but the specific mechanisim is not yet completely clear. In this study we cultured the myogenic satellite cell in vitro, carried out RNAi study in which smad3 is the target gene by RNAi. The influences of Smad3 gene interference on myogenic satellite cell proliferation were studied. The changes of MyoD and Myostatin gene after smad3 gene interference in myogenic satellite cell were also studied, and the relationship of regulation among three genes was further explained. Together, this results show that myostatin inhibits myogenic cell differentiation by down-regulating MyoD expression, but this inhibition is mediated through Smad3.
To develop an oral vaccine against Helicobacter pylori infection, the H.pylori napA gene, encoding the neutrophil-activating protein (NAP), was expressed in a live delivery vehicle lactococcus lactis. And the immunogenicity of the recombinant NAP was evaluated after the lactococcus lactis was administrated orally into mice. First, the napA gene of Helicobacter pylori was cloned into the prokaryotic expressive vector pNICE:sec. Second, the recombinant vector pNICE:sec-nap was transformated into Lactococcus lactis strain NZ9000 to express NAP protein. Then the recombinant NAP was induced to express and was identified by SDS-PAGE and Western-bloting. Finally, ICR mice were randomly divided into 4 groups and administrated orally with PBS, L.lactis pNICE:sec, L.lactis pNICE:sec-nap, and the inactivated H.pylori respectively. The specific IgG and IgA were identified after 7 times immunization. The results we got in this experiment were described as follows. The napA was amplified and cloned in the vector pNICE:sec successfully. The fusion protein (17kDa) was expressed in L.lactis by the induction of the nisin. The quantity of expression accounted for 9.5% of the total bacterial protein. Western bolt analysis confirmed that fusion protein could be recognized specifically by the serum of anti-H.pylori. Anti-NAP IgG titers in the serum of L.lactis pNICE:sec-nap group was higher than the L.lactis pNICE:sec group and no obviously difference with the inactivated H.pylori. The anti-NAP IgA titer in the intestinal mucosa of L.lactis pNICE:sec-nap group was higher than other groups. These results suggest that the recombinant L.lactis which expresses napA, may be applicable as an oral vaccine to induce protective immunity against H.pylori.
To characterize the promoter and signal-peptide activity of expression vector pGPST, lacZ gene was inserted into vector pGPST yielding the recombinant plasmid pGPST-lacZ and it was translated into Bacillus subtilis AS.1700 by electroporation. The active of β-galactosidase in culture supernatant and bacteria were analyzed respectively. In culture supernatant the enzyme active reaches peak after 22h, approximately 26 mU/mL. then decrease slowly. In bacteria pellet the most expression was almost 6 mU/mL after cultured 14h. While none active was detected in negative control. The results show that the promoter and signal-peptide can be used to express heterologous gene in Bacillus subtilis. The study provides considerable data which are useful for the further development of the expression vector pGPST and the study of foreign gene expression in Bacillus subtilis. The vector pGPST will play a significant role in gene expression in Bacillus subtilis.
Retroviral expression system which consists of retroviral vector, envelop protein vector and packaging cell line is an efficient expression system for recombinant protein. It has great potential in gene therapy and biopharmacy. Transcriptional active genome regions are the preferred targets for retrovirus integration. Furthermore, VSV-G protein enables this system a broader host range and makes virus integration more efficient.After infection of high-titer virus,high production clones can be selected through simple screening. So far, the research on retrovirus expression system has developed into application in bio-pharmacy industry. Here we introduce the composition of this system and the mechanism of virus transduction and summarize the application and prospect of retroviral expression system.
Cecropin is a kind of heat-durable and broad-spectrum antibacterial polypeptides which has strong effect against bacteria, fungi, virus and some pathogenic microorganisms. Today Cecropin has been widely applied into plant genetic engineering，antiviral study, and inhibiting tumor cell proliferation. In this paper, we review its Structure-function relationship, antibacterial mechanism, and the application on transgenic plants for bacterium resistance. Expression of Cecropin in plants has a great application potential in bacterium resistance. Deep analyses and research of molecular structure and action mechanism can promote the transgenic plants antibacterial research.
Transformation-competent large DNA vector including the BIBAC(binary bacterial artificial chromosome) and TAC(transformation-competent artificial chromosome) can be used to clone Large DNA fragments effectively, and get transformants as well as relational gene’s information. In this paper, the categories, characters, recent progress and applications in genetic improvements on crop of the transformation-competent large DNA vectors were reviewed. By the way, the applying of transformation-competent large DNA vectors on peanut breeding using wide peanut species were prospected.
Microbial lipases have been widely used in traditional industries, as well as in emerging biocatalysed areas owing to their ability to catalyze a variety of reactions in aqueous and non-aqueous media. Therefore, it is very important to enhance amount of lipase production by recombinant overexpression for meeting market demand. This article presents a critical review of main and novel strategies which have been employed for recombinant expression of microbial lipases, including codon optimization, fusion and co-expression, dual expression system based on hybrid promoters, homologous overexpression, cell surface displaying and high-throughput screening based on gene library of expression. These new technologies are gradually coming to the forefront in the recombinant expression of lipase, especially for cell surface displaying and high-throughput screening based on gene library of expression. Meanwhile, several recombinant expressions for representative microbial lipases were also introduced and discussed, which are available for consultation when attempting to overexpress any lipases by scientists and industrialists.
Biosensor technology is a new -high powerful biological analytical technique, which incorporating a biological or biomimetic sensing element, either connected to or integrated within a transducer system, and harnessing the specificity, sensitivity, small and convenient for use characteristics, have developed rapidly in recent years, and it has been applied in many fields such as health care, food industry and veterinary and husbandry, and become one of the main research items. In this paper, the concept and principles of biosensor, the classification, the research and application in main fields of biosensor were reviewed, the commercial situation, the advantages and limitations of biosensor were addressed, and the prospect of development of biosensor was presented.
