Construction of a recombinant attenuated Salmonella typhimurium nucliec acid vaccine carrying Helicobacter pylori HpaA and detecton of its immunogenicity / 解放军医学杂志

ABSTRACT

Objective To construct a recombinant live attenuated Salmonella typhimurium nucleic acid vaccine carrying Helicobacter pylori ( H. pylori ) hpaA gene and to detect its immunogenicity. Methods hpaA gene fragment was amplified by polymerase chain reaction (PCR) from the genomic DNA of the standard H. pylori strain and cloned into pUCmT vector. DNA sequence of the amplified hpaA gene was assayed,then cloned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions. The recombinant plasmid was used to transform Escherichia coli DH5?,and the positive clones were screened with PCR reaction and restriction enzyme digestion. Then,the recombinant pIRES-hpaA was transformed to LB5000,then the recombinant plasmid was transformed to SL7207,and the recombinant strain was passaged repeatedly. Finally,recombinant pIRES-hpaA was transfected to COS-7 cells with Lipofectamine TM 2000. The immunogenicity of expressed hpaA protein was determined with SDS-PAGE Western blot. Results The hpaA gene fragment of the 750 base pair was amplified from the genomic DNA,and it was consistent with the sequence of the H. pylori hpaA by sequence analysis. It was confirmed by PCR and restriction enzyme digestion that Helicobacter pylori hpaA gene was inserted into the eukaryotic expression vector pIRES and a stable recombinant live attenuated Salmonella typhimurium nucleic acid vaccine carrying Helicobacter pylori hpaA gene was successfully constructed,and specific strip of hpaA expressed by pIRES-hpaA could be detected with Western blotting. Conclusion The recombinant attenuated Salmonella typhimurium nucleic acid vaccine strain expressing HpaA protein with immunogenicity was constructed,it might be helpful for further investigation concerning the immune action of the nucleic acid vaccine in vivo .