It was focused on that,the binding efficiency of Vascular Endothelial Growth Factor(VEGF)and Vascular Endothelial Growth Factor Receptor-2(VEGFR-2)competitively inhibited by bivalent single-chain Fv(BsFv),and BsFv's construction,expression and bioactivity identification. PCR primers were designed to introduce interlinker(G4S)3 between two scFv AK404 fragments,which were ligated with vector pET22b and transformed into E.coli BL21(DE3).The expression product was purified by IMAC and renatured by dialysis. The KDR-binding activity was determined by Bio-Layer Interferometry(BLI)and its influence on the propagation of HUVEC cell induced by VEGF. Bsfv was successfully constructed,and expressed as inclusion body after having been induced by 1 mmol/L IPTG for 6h at 37 ℃. After purification by IMAC and renaturation of the denatured recombinant protein,the BsFv with bioactivity and 90% purity was obtained. It was characterized to be target protein by Western blotting. The result of binding experiment in vitro based on BLI had proved that BsFv has higher binding ability to KDR3 than AK404. Additionally,BsFv was shown to inhibit the HUVEC cell's proliferation in a concentration-dependent manner,and the inhibition rate was relatively higher than that of AK404. All the results indicated that BsFv have higher anti-KDR activity compared with scFv,which provided a potential candidate drug in further research on its application on antiangiogenic oncotherapy and tumor diagnostics.
Miz1 is an important transcription factor,and also a signal-and pathway-specific modulator or regulator. Upon TNFα stimulation,Miz1 undergoes ubiquitination and degradation,releasing its inhibition on JNK signaling pathway,which leads to the activation of JNK. Phosphorylation and ubiquitination have multiple connections. Recent study showes that protein kinase Akt can specifically phosphorylate Miz1 to regulate cell-cycle arrest after DNA damage. Through the site-directed mutagenesis of Mus wild-type Miz1 at specifically phosphorylation site to get S419A Miz1,and then immunoblotting and in vivo ubiquitination assay,the results show that phosphorylation of Miz1 by Akt is not requied for its ubiquitination,and even suppresses it
To clone and identify the gene of porcine selenoprotein 15kDa(Sep15), Sep15 ,conduct the site-directed mutagenesis and prokaryotic expression for the further functional study of Sep15 using pig models. The total RNA was extracted from pig spleen for RT-PCR,and then the Sep15 cDNA sequence of 1230 bp containing the open reading frame(ORF)till to poly(A)tail was cloned. The 489 bp of ORF sequence had 85.1% identity to that of human,while their amino acid sequences had 92.7% identity. Like those in various mammals,the porcine Sep15 cDNA bore the common characteristics of an in-frame TGA for selenocysteine(Sec)and a Sec insertion sequence element of Form 2 located in the 3'-untranslated region. By inverse PCR,the TGA codon for Sec was mutated into TGC for cysteine(Cys). The mutant was recombined to pET30 vector and expressed in E. coli BL21(DE3)under the induction of 0.4 mmol/L IPTG for 3h. The expressed recombinant protein with His-tag detected by SDS-PAGE was about 23kDa. In Western blot assays,both the rabbit anti-sera generated by the purified recombinant protein and the commercial murine antibody against the peptide downstream the Sec residue of human Sep15 were able to detect the recombinant protein. In conclusion,the porcine Sep15 was cloned and identified for the first time,and its molecular characteristics shared high similarity with those of human SEP15 . And the Cys-mutant of porcine Sep15 cross reacted with the antibody against the C-terminal of human Sep15.
Objective:To evaluate the immune response of polypeptide vaccines Aβ1-15-PADRE(Aβ-T)against Alzheimer's disease containing the immunodominant B cell epitope from β-amyloid and pan-DR helper T cell epitopes and determine whether various adjuvants could boost the efficacy or performance of the vaccine in mouse model. Methods:The polypeptides of 2Aβ1-15-PADRE containing two B cell epitopes Aβ1-15 and one pan-DR helper T cell epitope PADRE was be artificially synthesized as polypeptide vaccines Aβ1-15-PADRE. Compared to the PBS control or untreated control,the immunogenicity of polypeptide vaccines without adjuvant and with four different adjuvants(Aluminum,Freund's adjuvant,MF59 adjuvant and Abisco adjuvant respectively)were evaluated in Balb/C mouse model. Results:All groups produced the specific antibody IgG against Aβ. But the four adjuvant groups were better to generate immune response than Mock group or negative control. Of these,the titer of antibodies produced by the Freund's adjuvant group was highest. The results of dot blotting showed that the sera antibodies could bind to the oligomer of Aβ better than the monomer form. But the antibodies have not binding reaction to the fiber form. Conclusion:All of these adjuvants could enhance the effect of polypeptide vaccine against Alzheimer's disease in mouse model. All of the sera antibodies bind to the oligomer form of Aβ. The immunological character shows that the polypeptide vaccine is potential in clinical trial.
AtNHXS1 ,a new vacuolar Na+/H+antiporter gene which was obtained from Arabidopsis thaliana AtNHX1 gene by DNA shuffling technology,was transformed into tobacco by Agrobacterium-mediated leaf disc. After hygromycin screened and PCR test,ten transgenic T2 lines were identified,and two lines of which were single gene insertion were used for further salt tolerance assays. Real-time PCR analyses showed that the expression levels of AtNHXS1 mRNA were significantly higher after salt treatment among different tissues,especially in leaves in transgenic lines. After salt stress,the growth status of transgenic lines were dominant better than the wild type lines. Under the treatment of 400 mmol/L NaCl,the transgenic plants grew well but the wild type plants almost nearly died. The result suggested that the transgenic AtNHXS1 tobacco could significantly improve the ability of plant salt tolerance.
To construct cloning vector pMDNos,terminator Nos was amplified from the template pBI121 plasmid by PCR. pMDKN vector was formed by combination between pMDNos fragment and KSTI3 from plasmid pMDKSTI3 by restriction enzyme digestion. Finally,promoter CaMV35S and chloramphenicol resistance gene Cat cloned from pCAMBIA2201 were all introduced to the vector pMDKN. DNA sequencing result showed that KSTI3 gene,promoter CaMV35S ,terminator Nos and Cat gene were completely consistent with the original sequence,so new eukaryotic expression vector pMDCKN-Cat was successfully constructed. pMDCKN-Cat was transformed into Dunaliella salina ( D. salina )with LiAc/PEG method,and recombinant D. salina was selected under chloramphenicol pressure and by PCR detection. Western blotting ultimately showed that a clear strip with the molecular weight of 20.1kDa appeared on the nitrocellulose membrane,and proved that trypsin inhibitor KSTI3 was expressed in D. salina cells.
Objective:To achieve surface display of H.pylori ure B fusion epitope by vector pHIE3N. Methods:Multiple cloning site was introduced into pHIE3N in order to express the H.pylori ure B fusion epitope protein. The recombinant strain was confirmed by SDS-PAGE,Western blot and ELISA. Result:The inserted sequence of the reconstructed plasmid was correct. The molecular weight of the fused Ure B protein was about 60 kDa which had proved by SDS-PAGE and Western blot analysis,ELISA results showed that the Ure B fusion epitope expressed on cell surfaces. Conclusion:The Ure B fusion epitope was successfully expressed on the sruface of E.coli DH5α using pHIE3N surface display vector. This result will contribute to the vaccine research of H.pylori.
Methanobactin(mb)is biochelator that appears to function as an agent for copper acquisition and uptake in methanotrophs. The production of mb during growth of Methylosinus trichosporium IMV 3011 on methane and methanol was determined by split nitrate mineral salts/CAS-Cu(chrome azurol S)plates. mb was found to be accumulated in the spent medium of Methylosinus trichosporium IMV 3011 under copper-limited condition. The mb production ability of methanol-grown cells were obviously higher than that of methane-grown cells. It has been speculated that methanol can act as the electron-donating substrate to regenerate the NADH and drive mb synthesis. The effect of mb from Methylosinus trichosporium IMV3011 on the growth lag,growth rate and whole-cell pMMO(particles Methane Monooxygenase)activity of other methanotrophs were measured respectively to assess the specifity of mb. It has been shown that mb from Methylosinus trichosporium IMV 3011 can accelerate the expression of whole cell pMMO activity of other methanotrophs when cultures are exposed to elevatory copper concentration. Also,mb from Methylosinus trichosporium IMV 3011 can shorten the growth lag and increase the growth rate of other methanotrophs either in elevatory or constant copper concentration. The results suggested that other methanotroph may take delivery of copper from mb released by Methylosinus trichosporium IMV 3011.
The accumulation of coproporphyrin Ⅲ,which is byproduct generated in vitamin B12 fermentation of Pseudomonas denitrificans,greatly affect the biosynthesis of vitamin B12 and the extraction in industrial production. An effective method for determining coproporphyrin Ⅲ concentration directly after suitable treatment of the fermentation broth. Furthermore,the influence of oxygen supply levels,carbon dioxide concentrations and pH on coproporphyrin Ⅲ biosynthesis were investigated,the optimal control strategy was implemented in 120 m3 industrial bioreactor. The results revealed that higher oxygen supply promoted the coproporphyrin Ⅲ accumulation,controlling the inlet carbon dioxide concentration at 8.6±0.8% and pH at 7.0±0.12 could greatly inhibit the coproporphyrin Ⅲ biosynthesis,and the vitamin B12 production increased for 15% than that of control.
The application of polyacrylonitrile fibers surface modification by Corynebacterium nitrilophilus NHase was studied through capillary effect, scanning electron microscopy and dyeing experiments. The results indicated that C. nitrilophilus NHase was effective in surface modification of polyacrylonitrile fibers,which enhanced wettability and dyeing ability of polyacrylonitrile fibers by 43.5% and 85.7%.In order to improve the production of C. nitrilophilus NHase,Single-factor experiments and Orthogonal experiment were used to optimize the culture condition in shake flakes. The optimized results were as follows:15 g/L glucose was used as carbon source;Nitrogen was the combination of yeast extract and ammonium chloride,whose concentration was 3 g/L,1 g/L respectively;The optimal concentration of inducer urea was 10 g/L ;The concentration of cofactor cobalt was 0.07 g/L. The NHase activity reached 45.7 U/ml compared with the initial activity of 16.2 U/ ml,which was 2.8 times after optimization in shake flakes.
The biofilms make it more difficult to treat the diseases caused by pathogens with antibiotics. A kind of diketopiperazine(DKP)——cyclo(Pro-Phe)were found can inhibit the biofilms formation of Staphylococcus aureus,Pseudomonas aeruginosa and Candida albicans. The results of crystal violet staining,CFU(colony forming unit)analysis,and the structure analysis by optical microscope and atomic force microscope indicated that the biofilms of S. aureus and P. aeruginosa were almost disappeared with 10 mg/ml DKP,and the biofilms of C. albicans was significantly inhibited by 12 mg/ml DKP. This brings hope to the research work of novel biofilm inhibitors and cure of the biofilms-associated infections.
Recombinant human α-lactalbumin(rHLA)could be expressed in mammary gland bioreactor effectively. However,it is very difficult to purify the target protein. Primary proteins’ properties,such as surface hydrophobicity and electric potential,were compared through molecular simulation and calculation,based on which a fast purification process with high resolution was designed. Immunoglobulin G,which would disturb chromatographic purification process,was firstly removed using ammonium sulfate precipitation with orthogonal optimization. Then the subsequent hydrophobic interaction chromatography(HIC)became more stable and rHLA with 95% purity was obtained through separating successfully from its homologous courterpart,i.e.,bovine α-lactalbumin in HIC. The overall recovery of rHLA was up to 48.6%. Activity detection and circular dichroism spectroscopy(CD)confirmed the regulatory activity for β-1,4-galactosyltransferase and native structure of purified rHLA.
The genes of adhB and pdc,coding ADHII and PDC respectively,are key genes of alcohol production pathway in Zymomonas mobilis.The following vectors,pUC-adhB,pUC-pdc and pUC-adhB-pdc,were created by inserting adhB and pdc into the plasmid pUC18. Both adhB and pdc have a ribosome binding site of rbcLS of Synechococcus sp.PCC7942. The results of biological activity detections showed that ribosome binding sites of rbcLS of PCC7942 can effectively induce the expression of adhB and pdc in E.coli. Jar fermentation showed that the recombined E.coli's ability of ethanol production was obviously improved compared with the initial strain. Aldehyde indicator plate was displaced by a new method for Biological activity detection because of its defects.The new method was as follows. Induced expression of the genes by IPTG,add substrates for the enzymes ADHII and PDC,cultivate for 0.5h to 1h at 37℃ and then add Schiff reagent.The results show that the improved method is more simple and reliable than Aldehyde indicator plate.
The suicide plasmid pUC-ldhD-Ter containing homologous sequence from Lacbacillus rhamnosus was transformed into Lactobacillus rhamnosus JCM 1553,Two recombinant stains have been constructed successfully and named GL-1,GL-2. PCR was applied to clone the gene of tetracycline from the chromosomal DNA of recombinant stains. The growth curve of the recombinant Lacbacillus rhamnosus was consistent with that of the wild-type strain.The highest yield of L-lactic acid in Erlenmeyer flasks from 10% glucose at 37℃/200 r/min by GL-1 and GL-2 are 93.956 g/L,and 93.693 g/L after 40h.The conversion of glucose are 94.82% and 94.56%. OD600 value of the two strains achieve 25.49 and 24.66.The residual sugar content are 0.60% and 0.63% respectively.The result showed no remarkable difference from the wild type.
The objective gene fragments cowpea trypsin gene CPTI,35S promoter,NOS terminator and cotton endogenous reference gene Sad1were cloned by PCR method and ligated into cloning vector to construct a standard reference plasmid pGB, When pGB,transgenic cotton and non-transgenic cotton DNA were used as template to amplify CPTI,35S,NOS and Sad1 gene,four targets DNA fragments can be amplified from pGB and transgnic cotton,but only SadI can be amplified from non-transgenic cotton. It was draw that quantitative standard curve of CPTI,35S,NOS and Sad1 gene by quantitative PCR method. The correlation coefficient of the standard curve reached more than 0.985,indicating that the quantitative PCR reaction system has a good relativity between Ct value and the initial concentration. Also,quantitative PCR reaction system has reproducibility and stability,can be used for quantitative analysis of real samples.
Objectives:To improve the expression of gene of interest in a lentiviral vector with complicated gene distribution. Methods:Lentiviruses have complicated gene distributions such as overlapping Env,Rre,Rev and Tat on their genomes. To avoid damaging the reproduction efficiency of these vectors,it is better to retain as much as possible the original gene structure when replacing the unwanted Env gene with a gene of interest. A reporter gene luc was first inserted into the lentiviral vector in place of the Env gene. The resultant plasmid did not express detectable luciferase activity. In order to improve the target gene expression, a frameshift mutation and a stop codon upstream the luc gene were introduced. Results:After the introduction of the upstream frameshift mutation and a stop codon,the luciferase expression was greatly enhanced. Conclusion:By introducing frameshift mutation and stop codon in the upstream reading frame, the expression of the downstream open reading frame could be largely increased.
Intein-based protein cleavage can be useful tools of recombinant protein purification,ligation and cyclization. However,existing methods were often complicated by low cleavage efficiency,spontaneous cleavages,and low yield of desired protein products. Site-specific cleavages at the N-or C-terminus of an intein can result from alterations of the protein splicing reaction. In N-terminal cleavage,the intein's last residue is mutated to abolish step 3 of the splicing mechanism. Step 1 of the splicing mechanism can still happen,and the resulting ester bond can spontaneously hydrolyze to separate the N-extein from the intein. In C-cleavage,the intein's first residue is mutated to abolish step 1 of the splicing mechanism,but Asn cyclization can still occur to break the peptide bond between the C-extein and intein. The first residue(Cys)or last residue(Asn)mutated split-inteins through site-specific mutagenesis using high splicing S1 and S11 Ssp GyrB split-inteins were constructed and their cleavage efficiency in E.coli cells were tested. Splicing was blocked by site-specific mutation,N-cleavage of S11 split-intein and C-cleavage of S1 split-intein were increased in E.coli cells. This will help us to investigate controllable protein cleavage in vitro,further establish efficient protein purification system and investigate splicing mechanism of split-inteins.
In order to study its specificities,the NotⅠR gene was cloned from Nocardia otitidis-caviarum. Firstly,the genome DNA of Enterobacter agglomerans was extracted as template,obtained EagⅠmethylase gene by PCR and connected EagⅠM gene to pBR322 vector to gain recombinant expression plasmid pBR322-Eag ⅠM. Then transformed this plasmid into E. coli 2566. Secondly,extracted the genome DNA from Nocardia otitidis-caviarum as template and obtained the restriction enzyme NotⅠR gene by PCR. After ligating the NotⅠR gene to pACYC184-PT7,the pACYC184-PT7-NotⅠR plasmid was transformed into the ER2566 which was protected through the methylation by pBR322-EagⅠM recombinant plasmid. The engineered strain ER2566 could be induced to express restriction enzymes NotⅠ by IPTG and the induction conditions were optimized to make its expression mostly in soluble form. The enzyme was purified by ÄKTA purifier 100 protein purification system. Through DEAE Sephrose FF,phenyl HP and Superdex 75 10/300 GL molecular sieve chromatography,the Not Ⅰenzyme was purified 35-fold,the yield was 9.8 × 106 Units / g wet cell which was up to17.8% of the crude enzyme and the specific activity of the purified NotⅠwas 1.37 × 106U/mg. Digestion results showed that the enzyme was purified to homogeneity and was free of detectable contamination by other DNase(exo and endo). After optimization of the expression and purification conditions,the yield and efficiency of NotⅠ enzyme were greatly improved in comparison with that previously reported.
Recombinant antibodies, especially ScFv fragments, can be applied as detection reagents and even substitute for some reagents used in immunoassays such as antibody-enzyme conjugates. For ScFv fragments, a universal system available is necessary. A vector system was constructed based on pPICZα/Fc, in which the hinge, CH2 and CH3 domains (Fc fragment) of human IgG1 and His-tag were cloned into the Pichia expression vector pPICZα. Two fragments of ScFv were introduced into pPICZα/Fc, which can bind HBsAg and rabies virus antigen, to yield the expression cassette pPICZα/ScFv-Fc. Following fermentation in a 1-liter reactor, the fusions were expressed at high levels in the methylotrophic yeast Pichia pastoris, secreted as dimeric forms in the culture, and purified by protein A column chromatography. The expression yield can reach 20~30mg/L of culture medium. The ScFv-Fc fusion proteins retain the biological binding ability of the parent ScFv. Furthermore, the successful expression and maintenance of the binding activity verify the efficacy of the vector system for use as detection reagents in vitro, by reacting with the specific antigens and being readily detected using general anti-human IgG antibodies.
Stem cells research and the technology of stem cell-based regenerative medicine have become one of the important parts for the development of biomedicines. The research and development of stem cell and regenerative medicine will have important impacts on the medical market for the patients with tissue and/or organ deficits or dysfunction. Based on the origin and the way obtained,stem cells are classcified as embryonic stem cells(ESCs),induced pluripotent stem cells(iPSCs)and adult stem cells(ASCs). Different types of stem cells may have different characteristics,and different risk potential for clinical use. The products of stem cells may have different clinical indication,and be reviewed by different standards. The current progress in the clinical application and management strategies of stem cells were reviewed
Differentiated somatic cells can be reprogramed to induced pluripotent stem cells(iPS cells)by thansfection of a combination of specific genes,which is the most notable stem cell manufacturing technology in the stem cell research field in recent years.iPS cell not only is new model for the study of reprogramming mechanism of somatic cell,but also bring a new hope for the research of disease development mechanism and specific cell therapy. The preparation methods of iPS cells and related factors that influence transformation rate of iPS cells and pluripotency maintenance were summarized.
White spot syndrome virus was deeply studied at all times since the white spot syndrome disease break out in shrimp culture from 1990’. The functional research of the WSSV structural proteins,especially the virus membrane proteins,and acquired the notable body protection for shrimp was mainly focused on. A comprehensive summary of the research progress of the WSSV membrane proteins,including utilized the membrane proteins as a subunit vaccine;utilized the corresponding antibodies of membrane proteins;constructed the nucleic acid vaccine;and utilized the RNAi technology for shrimp protection was given;and a view of the future application of WSSV membrane proteins in prevent of the white spot syndrome diseases was also provided.
The budding yeast,Saccharomyces cerevisiae,has been used as eukaryotic model organism in "ome" level research. "Omics" technologies were mainly composed of genomics,transcriptomics,proteomics and metabolomics. The recent advancements of "ome" level research in yeast were surveyed,then their application in strain improvement by genetic engineering was discussed,including the industrial yeast strains in bioethanol and winemaking processes.
With the development of biotechnology,The contradiction between developing transgenic organism and public concerns in its security is very serious. Through analyzing public sentiment to transgenic biotechnology in China,a conclusion is drawn that the insufficience of transgenic biotechnology risk communication in China is those objective is not clear,information disclosure is insufficient,the form and content of risk communication is dry,and public institutions fail to play their role. On this basis,suggestions for strengthening risk communication in transgenic biotechnology are put forward:to clear the object of risk communication;to establish the coordination framework of risk communication;to build a platform for information publicity and information monitoring;to improve the role of administrative services and public institutions in risk communication;to reform the policy of scientific project.
