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《Chinese Journal of Microecology》 2008-06
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Gene cloning and expression of VacA-ctxB recombinant protein to Helicobacter pylori

ZHANG Ren-fei1,YANG Zhi-bang1,LI Jun-jie1,XIA Li-jun2,LI Chang-qing3,CHEN Pu4(1.Department of Pathobiology,Chongqing University of Medical Sciences,Chongqing 400016,China;2.Department of Clinical Laboratory,Nuclear Industry 416 Hospital,Chengdu 610051,China;3.Department of Clinical Laboratory,the 6th People′s Hospital of Chengdu,Chengdu 610051,China;4.Department of Clinical Labortory,the 1st Affiliated Hospital of Chongqing University of Medical Science,Chongqing 400022,China)  
Objective To lay a foundation for preparation of prophylaxis vaccine and therapy of H.pylori infection,the fusion gene of H.pylori vacA and cholera toxin subunit B(ctxB) was constructed and VCTB recombinant protein was expressed.Methods The vacA toxic subunit gene was amplified using PCR and template of DNA genome from H.pylori,and cloned into plasmid pQE30 to acquire plasmid pQE30-vacA.The ctxB gene was amplified,using pET32(a)+-ctxB as the template and inserted into pQE30-vacA to construct the expressing plasmid pQE-vctB containing vacA and ctxB genes.pQE-vctB was cloned into E.coli Top10,then translated into E.coli DH5α to be induced and expressed.VCTB recombinant protein was analyzed by SDS-PAGE after purified by Ni2+-NAT chromatography.Its antigenicit`y was identified by Western blot.To confirm its immunogenicity,the antibodies of VacA and CtxB in the serum were detected by ELISA after immunizing by the VCTB recombinant protein.Results The vacA gene sequenced as about 723 bp and ctxB gene sequenced as about 372 bp were consistent with the anticipated length of DNA.The vctB fusion gene sequenced as 1092 bp by the sequencing was in conformit with the genebank,and encoded polypeptides of 364 amino acid residues.The expression product was about 20% of total cell protein,and its relative molecular weight was 40 000,consistent with the anticipated weight analysed by SDS-PAGE.A single strap was clear after recombinant protein was puritied and the purity of recombinant protein was above 92% by SDS-PAGE.It was confirmed that VCTB recombinant protein could react specifically with VacA antibody in human serum identified by Western blot and with CtxB antibody in rabbit serum detected by ELISA.Conclusion The vector containing vacA and ctxB fusion gene of H.pylori was constructed successfully,and VCTB recombinant protein was expressed in E.coli DH5α.VCTB fusion protein expressed can be used to prepare oral vaccine for their better antigenicity and immunogenicity.
【CateGory Index】: R392.33
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