Thymosin α1 (Tα1), an immune booster, plays critical roles in the maturation, differentiation and function of T-cells. Tα1 mainly was used to cure various diseases of immunodeficiency and virus infection in clinic, such as hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), cancers and so on. Several reasons confine application of the Tα1 in clinic, which include the available thymus of calf or porcine for extraction of Tα1, higher cost to yield Tα1 by chemical synthesis method, and existing safety problems by traditional expression system such as Escherichia coli and transgenic animal using genetic engineering way, because of contamination of E.coil-derived endotoxin and some zoonotic pathogens. In order to meet clinical demand for Tα1, the plant-derived Tα1 was tentatively expressed in transgenic tobacco. The tα1 gene (124 bp) was designed and synthesized according to the plant codon usage bias and created a novel 4×tα1 concatemer (four copies of the tα1 gene arranged end-to-end in tandem, designated 4×tα1). Subsequently, a plant binary expression vector, p35s∷4×tα1 with twin T-DNAs was constructed and introduced into tobacco via Agrobacterium tumefaciens-mediated transformation. Fourteen independent transgenic tobacco plants were confirmed by PCR and Southern blot analysis, and target 4×tα1 gene and selective nptⅡ gene were integrated into tobacco genome at different copies or sites in T0 generation, respectively. In order to analyze the separation of 4×tα1 and nptⅡ, 177 transgenic tobacco plants was assayed with PCR in T1-generation, two transgenic tobacco plats which only carried 4×tα1 was obtained but without selective gene nptII. The ELISA results showed that content of the 4×Tα1 in transgenic tobacco plats ranged from 180.46 ng/g·fw (line 81) to 756.87ng/g·fw (line 86), and Western blot result confirmed that tobacco-derived 4×Tα1 possessed immunocompetence. The MTT ［3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide-experiment showed the 4×Tα1 protein derived from transgenic tobacco exhibited bioactivity that can stimulate the proliferation of mice splenic lymphocytes in vitro. The experimental data will provide significant reference to produce recombinant therapeutic proteins including Tα1 using safe plant expression system.
Objective: To construct eukaryotic expression vectors containing genes of chimeric intron and anti-bFGF antibody and express in 293T cells for exploring the effect of chimeric intron on antibody expression. Methods: Genes of chimeric intron and Fd、κ chain were amplified from vectors pCl-neo and pComb3-Fab25 respectively, then inserted in different combinations into expression vector pIgG containing human immunoglobulin IgG1 Fc region. Recombinant plasmids were transfected into 293T cells by liposome polyethyleneimine(PEI). Transfection result was observed by fluorescence microscope and antibody expression was analyzed by sandwich ELISA and Western blot. Results: The results of sequencing and restriction enzyme digestion analysis showed that intron and antibody genes were successfully inserted into expression vector pIgG. Fluorescence observations indicated that plasmids were transfected into 293T cells and antibody in culture supernatant was detected by Western blot. Antibody concentration of pIgG-Fd-κ、pIgG-Fd-intron-κ、pIgG-Fd-κ-intron and pIgG-Fd-intron-κ-intron was 1.21mg/L、0.468mg/L、7.39mg/L、0.601mg/L respectively. Conclusion: Recombinant expression vectors containing chimeric intron and antibody genes were constructed and expressed in 293T cells successfully and the chimeric intron may conduct important action. Antibody expression can be improved by inserting chimeric intron into 5’ upstream of κ chain, but depressed into 5’ upstream of Fd.
The non structural protein 1 (NSP1) of rotavirus (RV) plays a very important role in interaction between virus and host. In this article, the full-length gene of the NSP1 of strain TB-Chen was expressed in E.coli BL21(DE3) cells by using gene cloning and expression technologies, and immunogenicity, synthesis and subcellular distribution of the NSP1 protein in RV infected cells, and phylogenesis and genotypes of the NSP1 were studied. The results showed that the recombinant NSP1 protein (rNSP1) could be highly expressed in E.coli BL21(DE3) cells, which possesses 34.4% of total cell proteins. The rNSP1 could elicit specific antibodies in guinea pigs, the antibodies could react with the rNSP1 itself and NSP1 protein expressed in SA11 infected or Wa infected MA104 cells by Western blot and immunofluoresence assay. The results of immunofluoresence also showed that NSP1 protein was aggregated in the cytoplasm and formed radiate granular structure in SA11 infected MA104 cells but not in Wa infected MA104 cells. At least 16 recognized genotypes of NSP1 were classified in group A RVs, based on diversity of sequences of the NSP1 open reading frame, and TB-Chen strain NSP1 was defined as genotype A2. RVs with different NSP1 genotype have specific host ranges, RVs possessing the same NSP1 genotype can infect different species, the same species can be infected with RVs possessing different NSP1 genotypes. Genotypes A4 and A16 were only found in birds and only A4 and A16 were found in birds. The results obtained in the present study are important for further studying on structure, function and immunological properties of the NSP1.
Serine protease of hepatitis C virus, which is a key viral non-structural protein and anti-virus target, could degrade HCV polyprotein with cis and trans way to release viral function proteins. Objective To develop a method for evaluating the activities of serine protein or its inhibitors in vitro, a small recombinant peptide( named 2S) as a surrogate of natural substrate of HCV serine protease was designed and its specific sequence was refered to subtype 1a containing amino acid sequences of two linkage sites between HCV NS3 and NS4A, and NS4A and NS4B. A small peptide 2S was expressed finally in prokaryotic cells with GST fusion. Methods 2S gene was synthesized with PCR technique while bath BamH I and EcoR I restriction sites were inserted into 5’ and 3’ ends of the gene. The desired gene was enzymatically performed with BamH I and EcoR I and subcloned into pGEX-4T-2 vector. The recombinant plasmid pGEX-4T-2s was identified with sequencing after transformed into competent cells DH5α.The engineering bacteria was induced overnight by 0.5mM/L IPTG and GST-2S fusion protein was purified with GST affinity column and applied to be degraded with raw protease in phosphate buffer system in vitro. In addition, the catalytic characteristic was analyzed through SDS-PAGE electrophoresis and ELISA. Result The recombinant plasmid pGEX-4T-2s was successfully constructed and the desired protein purified was obtained by affinity column. The detection in PB catalytic system showed that GST-2S belt clearly disappeared with SDS-PAGE and A450 value of the test panel was much lower than positive control with ELISA. Conclusion A small recombinant peptide designed with two cutting sites of HCV serine protease could be degraded by the enzyme in an in vitro system and could be a surrogate as natural substrate HCV serine protease.
The CIRP gene of mouse was amplified from testis total RNA of BALB/C mouse by RT-PCR. PCR product was cloned into the pGEM-T vector to construct recombinant plasmid pGEM-CIRP for sequencing. The correct CIRP sequence was inserted into pQE-30-Xa to construct expression vector pQE-30-CIRP.It was transformed into host strain E.coli M15 and was induced by IPTG. It shows efficient expression in the E.coli by SDS-PAGE detected. The expression product exists in inclusion body. CIRP fusion protein was purified by Ni-NTA affinity chromatography and electroelution. The purified CIRP protein was used to immune BALB/C mice three times. Result of indirect ELISA shows that antibody titers of serum was 1∶105. The hybridoma technique was adopted to fusion. Positive hybridoma cell was screened by indirect ELISA and 3 times subcloning. Western blot was used to indentify McAb specificity. Indirect ELISA was used to identify McAb IgG subclasses and determine titer of McAb cell supernatants and ascites. 1A6, 2D3, 3C5 and 4C4 4 hybridoma cell line of stable secretion CIRP McAb was obtained. The antibody subclass of 1A6, 3C5, 4C4 were IgG2b equally, The antibody subclass of 2D3 was IgG3, light chains were κ chain equally. 4 monoclonal cell supernatant titers were more than 1∶103, ascites titer reaching more than 1∶107. Western blot analysis indicates four monoclonal antibodies can specifically combine with recombinant protein CIRP and had no response with void vector comparison. The ascites of cell line 3C5 was as primitive antibody to test natural CIRP expression status of a variety of cold treated organizations. It shows the ascites of cell line 3C5 can specifically recognize natural CIRP protein of heart, liver, spleen, lung, kidney, brain, testes and other tissues, and can be combined with CIRP McAb. This proves that the CIRP McAb has a good specificity and binding activity. McAb against CIRP will act as biological reagents to be used in study of the further research of CIRP functions and utilization.
Differential display reverse transcription PCR(DDRT-PCR) was used to identify differential expression genes in Beijing fatty chicken and Arbor Acres Broiler(AA) adipose tissue. A total of 10 ESTs were found using DDRT-PCR and reverse northern dot blot, all of 10 ESTs were compared with nucleotide sequences deposited in the nr database and Gallus database. XF2、YF1、YF2 and YF4 had their highly similar nucleotide sequences in nucleotide databases but with unknown functions. XF4 is similar to full-length cDNA clone CL0BA010ZF08 of Placenta of Homo sapiens, the identity is 83%; YF3 was found highly similar to MLL5 (LOC417712),but the function is unknow; XF5 and YF5 were highly similar to HMGN3; XF3 had no significant similarity with existing genes or ESTs and was regarded as new EST. The new EST was submitted to GenBank(Accession number: EU594549). The lays a foundation for further study on the mechanism of differential gene expression in Beijing fatty and AA adipose tissue.
The avermectin analog doramectin(CHC-B1), sold commercially as DectomexTM, was co-produced with the undesired analog CHC-B2, CHC-A1 and CHC-A2 by Streptomyces avermitilis bkd76-3 with the supplementation of cyclohexanecarboxylic acid(CHC) during fermentation. Gene deletion vector pXJ04(pKC1139::△aveD1+△aveD2) was used to delete aveD gene in S.avermitilis bkd76-3. The aveD gene was displaced by deletion allele on the plasmid via double crossover. Shaking flask experiments and HPLC analysis showed that the aveD deletion mutant no longer produced the undesired analogue CHC-A1 and CHC-A2, and only made two components which were confirmed as CHC-B1 and CHC-B2 by LC/MS analysis. The yield of CHC-B1 improved 78.19%, and B2 improved 602.3%, the ratio of effective component had a 93.16% increase. The deletion mutant was proved to be genetically stable, and thus might be promising strain in industrial production of doramectin.
PhoN gene that was amplified from Salmonella enterica serovar Typhimurium genomic DNA by PCR, was cloned into pMD18 T-Vector. Recombined transfer vector T-VectorphoN was digested by restriction enzymes of Spe I and Nde I, and then the purified phoN gene was inserted into shuttle vector pRADZ3 which was digested by the same restriction enzymes. The recombined shuttle vector pRADZ3phoN was identified by PCR and restriction analysis, and transformed into E.coli DH5α competent cell. A recombinant fusion PhoN protein was expressed in normal growth condition without induction. The expression of PhoN protein in E.coli DH5α was confirmed by Western blot. The U(Ⅵ) bioaccumulation performance of engineered E.coli was evaluated. The results showed that the maximum U(Ⅵ) bioaccumulation capacity of engineered E. coli increased four times compared to original E. coli, reaching 46.16 mg/g, and the removal rate of U(Ⅵ) was 92.32%.
Inverse-nest PCR was applied to clone the full sequence of some α-amylase from enrichment soil metagenomic DNA. The successfully achieved gene（GU045523）showed high homology (Identity:99%)with the partial coding sequence of acid-stable amylase from Bacillus sp. KR-8104. The putative mature peptide gene was inserted into pSE380, a recombinant plasmid pSE380-gxaa was constructed and transformed into Escherichia coli JM109. The purified amylase GXAA showed an optimal activity at pH 7.0 and 75℃,the Km value is 11.6g/L taking soluble starch as substrate. Mutants E27G,A450T and E27G-A450T were constructed, the property showed no remarkable difference from the wild type.
S-Adenosylmethionine (SAM) which is synthesized from methionine and ATP by S-adenosylmethionime synthetase (SAMS) plays an important role in many biological reactions. SAMS gene which was cloned from the genome of E.coli K12,and the recombinant E.coli JM109(pBR322-SAMS) strain which can constitutively express SAMS was constructed. The productivity of SAMS reached 1 176μ/L, and was 20% of total soluble proteins of recombinant strain. After 20%~40% ammonium sulfate fractionation, the solution was loaded on Phenyl-Sepharose Fast Flow column. The enzyme fraction was absorbed on the column and was eluted as concentration of ammonium sulfate decreased to 0. Subsequently the effluent wad loaded on Q-Sepharose Fast Flow column, and the enzyme was eluted as concentration of KCl increased to 0.3mol/L. After ammonium sulfate fractionation and two column chromatography, the enzyme was enriched 5 times with a 62% activity recovery. The purified enzyme had a specific activity of 48.7μ/mg protein. The purity of SAMS reached 92%. The optimum reactive pH was 8.5, and the recombinant enzyme activity changed little when incubated in the buffer of pH 7.5 on 4℃ for 10 h. The optimum reactive temperature of recombinant enzyme was 55℃, and the recombinant enzyme was more stable on the temperature of 20~35℃. KmL-Met of recombinant SAMS was 0.22mmol/L and VmaxL-Met was 1.07mmol/( L·h). KmATP was 0.52mmol/L and VmaxATP was 1.05mmol/( L·h)。
The medium and condition of liquid fermentation for the yield of lipase of Burkholderia sp. SYBC LIP-Y were optimized with single factor experiment and orthogonal design. The most suitable medium was formulated with the ingredients: 10 g/L tragantine, 15 g/L beef extract, 0.252 g/L NaNO3, 40 ml/L olive oil, 10 ml/L Triton x-100, pH 7.5, and 10% (V/V) of the inoculum size. The lipase activity reached 83.85 U/ml, which was 3.63 folds of that before optimization. Properties of the lipase after purification by aqueous two-phase system was studied. The optimum pH and temperature of the purified enzyme were 10.0 and 30℃ respectively. About 80% of the original activity is maintained by heating at 40℃ for 60 minutes. The lipase belonged to the cold-active lipase. Good temperature stability and resistant to alcohol were proved. There had wide application fields that can be believed.
The condition of both expression and purification of SUMO human epidermal growth factor in E.coli is discussed. After transforming recombinant expression vector into E.coli, the experiment optimizes the induction expression, and analysis through ion exchange chromatography NiNTA affinity chromatography and molecular sieve chromatography, adopting arabinose as the IPTG. The results are as follows: 37℃ is the most favorable temperature for induction expression of SUMO-hEGF in BL21(AI), 5.0g/L the most best concentration of arabinose, and 4h the best time for induction expression. Meanwhile, the quantity of expression is around 20.2%. The conclusion that hEGF is the purified protein is verified by Western blot.The good foundation for the development of hEGF genetic medicine is founded.
The threonine genetic engineering strains were constructed by a strategy of cut off branch metabolic pathways and overexpression thr operon in E.coli ITHR. The metA and ilvA genes were knocked out by Red-recombination systems and E.coli ITHR△metA、ITHR△ilvA and ITHR△metA△ilvA were constructed respectively. The pWYE065 containing thr operon were transformed into three mutant strains by electroporation. Red-batch cultures of genetic engineering strains were carried out in 5 L fermentors and the threonine concentration was determined by HPLC. The results showed that E.coli ITHR carrying pWYE065 could accumulated 5.55±0.51 g/L threonine. When metA and ilvA were knocked out respectively, the threonine productions reached 9.77±1.83 g/L and 8.65±1.42 g/L. Both metA and ilvA were knocked out simultaneously, the L-threonine production were increased to 13.6±1.14 g/L. The productivity of L-threonine by E.coli ITHR can be enhanced through the knocking out the gene of the key enzymes in branch metabolic pathways, including the methionine and isoleucine synthesis pathway.
A temperature sensitive plasmid-based procedure without linear DNA was developed for a rapid deletion of chromosomal target genes in Salmonella. To construct a recombinant vector, homologous regions and kana cassette with two FRT sites were amplified using corresponding templates and cloned into Ts-plasmid pHY304. The produced shuttle vector was then transformed into S.ty2. Under the pressure of temperature and antibiotic, homologous recombination occurred between the vector and genome of the host bacterium and the recombinants were selected by agar media with kanamycin. To delete kan resistant cassette, plasmid pCP20 was introduced into the recombinant. The markerless mutant strains were detected by genome PCR. The ssaV gene was successfully deleted by the modified method. A novel Ts-plasmid-based method has been established, which could be used for deletion of gene in other Gram-negative bacteria.
SYBR GreenⅠ real-time PCR was developed to determine the copy number of exogenous pta gene in transgenic wheat. A conserved wheat housekeeping gene, wx012, was used as an internal control and 5 times diluted non-transgenic wheat genome DNA is used as an initial template copy of wx012.So the y=-0.2667x+6.98, the standard curves of the cycle threshold (CT ) relative to the log of each initial template copy of wx012 was obtained. In the same way, the standard curves of pta y=-0.2118x+4.53 is obtained using 5 times diluted plasmid DNA which contains pta gene. By SYBR GreenⅠ real-time PCR the CT values of wx012 and pta gene in each transgenic wheat was got, the transgene copy number is calculated by comparing the initial template copy of pta to wx012 gene in the same transgenic wheat. The conclusion is, among the seven PCR putative transgenic plants: one was non-transgenic plant，one had one copy number，one had two copies，and the other had three，four copies，respectively.
The emergence of drug-resistant variants during antiretroviral therapy is a serious obstacle to sustained suppression of the human immunodeficiency virus type 1 (HIV-1). For that reason, continued drug discovery and resistance assays are essential for the treatment of HIV-1 infection. Using pseudovirus systems is a more attractive option for the screening of anti-HIV-1 drugs and the analysing of drug resistance. The construction and characterization of the pseudotyped HIV with a single-round infectivity and some novel cell-based pharmacological models for anti-HIV-1 compounds screening and drug-resistance analyzing by using pseudovirus systems were introduced. Meanwhile, a large number of studies have been summarized to prove that using these systems is accurate, safe and efficient.
Thaumatin-like proteins (TLPs) are a kind of plant defense proteins with many biological activities and functions. TLPs belong to the fifth grou Pof pathogenesis-related proteins. There are many researches on antifungal activity of TLPs in recent years. TLPs exhibit the glucanase activity, and they can bind and digest β-1,3 glucan which is a major component of fungal cell wall.In the crystal structure of TLPs, an acidic cleft on the surface of the protein appears to be necessary for antifungal activity. The relation between the structure and function, biological characteristics, and application in transgenic engineering about TLPs were reviewed
Cyanobacteria are used as a host of gene engineering to express foreign gene, their unique properties gradually attracting scientists' attention. The stable cyanobacteria transformation system has been constructed since 1990. In the past 20 years, more than 30 foreign genes have been successfully expressed in cyanobacteria. With the further investigation, cyanobacteria transgenic expression system has the potential to be extensively used for production of recombinant drugs, clean energy resources, farm chemical and for preventing the environment pollution et al. This review summed up the development of cyanobacteria genetic engineering and their uses in medicine, environment, agriculture and biosensor. Then, this review analyzed the development bottlenecks of the cyanobacteria transgenic engineering, the low expression rate of the foreign gene in cyanobacteria. Finally，it summarized the efforts that many scientists have made to improve the low expression rate, including: choosing high efficient gene and oral preparation, optimize transcription and translation component, adjust the host cell`s physiological state, et al.
Gasification of coal, oil, biomass or organic wastes generates synthesis gas, which consists primarily of CO, H2 and CO2. Synthesis gas may be used as substrates by some anaerobic bacteria to produce liquid fuels and chemicals such as ethanol, acetic acid, butanol and butyric acid. Anaerobic fermentation of synthesis gas is considered a promising and competing technology due to its advantages over catalytic techniques, and it's expected that synthesis gas fermentation will play a role in the conversion of biomass and organic wastes. Research progress in production of organic acids and alcohols via synthesis gas fermentation was reviewed, focusing on microorganisms, the metabolic pathway, key enzymes (especially carbon monoxide dehydrogenase /acetyl-CoA synthase) and bioreactors. Suggestions were also given to indicate areas where advances can be made.
Since the first approved lipopeptide antibiotics-datomycin launched in 2003, there were increasing indications and bright market prospects of daptomycin, resulting its manufacturing process and structure modification has become the focus of anti-infection drugs research and development.The course of leading compound A21978C and reviewed the evolution of synthetic techniques was traced. Moreover, manufacturing technology, biosynthetic gene clusters of daptomycin, and application of combinatorial biosynthesis in structure of daptomycin were also introduced emphatically.
