Construction,expression and identification of bivalent vaccine candidate antigens of human Helicobacter pylori

Aim To construct,identify a recombinant vector containing gene encoding heat shock protein A and VacA with Mr 13 000 and 26 000 simultaneously from human Hp.The recombinant vector be expressed in E.coli BL21,as well as analysed its antigenic for the exploiting vaccine of Hp.Methods The target genes encoding HspA and VacaA were amplified from Hp chromosome by PCR.And then digested by restricted endonuclease enzyme respectively,and inserted into the prokaryotic expression vector pET32a(+)digested by corresponding restricted endonuclease enzyme.The recombinant vectors pET32a(+)/HspA and pET32a(+)/VacA were used to select and transform for sequence analysis.After recombinant vector pET32a(+)/HspA and pET32a(+)/VacA digested by restricted endonuclease enzyme of Xhol,BamH Ⅰ simultanously,the pET32a(+)/HspA and VacA were extracted out of agarose electrophoresis,and connected by T4 ligase again.The recombinant vector pET32a(+)/HspA/VacA was used to select and transform,meanwhile expressed in E.coli BL21.The antigenic of recombinant fusion protein was analysed by western blot.Results Enzyme digestion analysis and sequencing showed that the target genes was found to be 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.SDS-PAGE analysis showed that recombinant vector could be expressed in E.coli BL21,its relative molecule mass(Mr)of expressed product was 59×103,while Mr of protein expressed by pET32a(+)was about 20×103,and soluble expression product accounted for 18.96 % of total bacterial protein.After purification with Ni 2+-NTA agarose resion,the purity of recombinant fusion protein was about 95%.The western blot result showed that recombinant fusion protein could be recognized by anti-Hp positive serum,suggesting that this protein had good antigenicity.Conclusion The genes coding HspA and VacA with Mr 13 000 and 26 000 respectively were cloned and expressed successfully.The results obtained lay the foundation for research on development of Hp protein and DNA vaccine and a quickly diagnostic kit applying to detection of Hp infection.