Cloning and onstruction of recombinant vector of bivalent vaccine candidate of human Helicobacter pylori / 重庆医科大学学报

ABSTRACT

Objective:To construct a recombinant vector containing gene encoding heat shock protein A (HspA) and VacA with Mr 13 000 and 26 000 simultaneously from human Helicobacter pylori(Hp)for study on expression in E.coli BL21,and analysis of their antigenicity.Methods:The target genes encoding HspA and VacaA were amplified from Hp chromosome by PCR,digested by restricted endonuclease enzyme respectively,and inserted into the corresponding enzyme digested prokaryotic expression vector pET32a(+).The recombinant vectors pET32a(+)/HspA and pET32a(+)/VacA were used to select and transform for sequence analysis.After recombinant vectors digested by restricted enzyme of Xhol,BamH Ⅰ simultaneously,the pET32a(+)/HspA and VacA were taken out of agarose electrophoresis,and connected by T4 ligase again.The recombinant vector pET32a(+)/HspA-VacA was used to select and identified by PCR and restricted endonuclease enzyme.Results:Enzyme digestion analysis and sequencing showed that the target genes were found in 1080 base pairs,and had been inserted into recombinant vector,but as compared with gene reported by GenBank,3.40% of the gene mutation and 1.11% of amino acid residues change in Hp happened respectively.Conclusion:The genes coding HspA and VacA with Mr 13 000 and 26 000 respectively are cloned successfully.The results obtained lay the foundation for research on development of Hp protein and DNA vaccine applying to prevention of Hp infection.