Construction of Helicobacter pylori hpaA nucleic acid vaccine and detection of its immunogenicity / 中华消化杂志

ABSTRACT

Objective To construct nucleic acid vaccine encoding Helicobacter pylori (H. pylori) hpaA gene. Methods The genomic DNA of the standard H. pylori strain 17874 was isolated. hpaA gene fragment was amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. The sequence of the amplified hpaA gene was assayed, and then cloned into the eukaryotic expression vector pIRES through a series of enzyme digestion and ligation reactions. The recombinant plasmid was transformed into competent Escherichia coli cells DH5?, and the positive clones were screened by PCR reaction and restriction enzyme digestion . Recombinant pIRES hpaA was transfected into COS 7 cells using Lipofectamine TM 2000, and the immunogenicity of expressed HpaA protein was detected with SDS PAGE and Western blot. Results The 750 base pair hpaA gene fragment was amplified from the genomic DNA and was consistant with the sequence of the H. pylori hpaA by sequence analysis. PCR and restriction enzyme digestion results revealed that the H. pylori hpaA gene was inserted into the eukaryotic expression vector pIRES, suggesting the nucleic acid vaccine pIRES hpaA was successfully constructed. The specific protein strip of HpaA expressed by pIRES hpaA was detected with Western blot. Conclusion The nucleic acid vaccine pIRES hpaA was successfully constructed which may help the further investigation towards the immune action of the nucleic acid vaccine.