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目的构建含幽门螺杆菌(Hp)hpaA基因的核酸疫苗。方法抽提Hp标准菌株CCUG17874基因组DNA,应用聚合酶链式反应(PCR)技术从基因组DNA扩增hpaA基因,克隆入pUCmT载体,检测hpaA基因序列,经过一系列酶切、连接反应将其克隆入真核表达载体pIRES,转入大肠杆菌,筛选阳性克隆,通过PCR和酶切反应鉴定。通过脂质体法将构建好的重组载体pIREShpaA转染COS7细胞,SDSPAGE及Western印迹法检测pIREShpaA表达HpaA蛋白的免疫原性。结果成功扩增出长约750bp的hpaA基因,测序结果表明扩增出的hpaA基因与HphpaA序列一致,PCR和酶切鉴定结果证实hpaA基因克隆入真核表达载体pIRES,成功构建了含hpaA基因的Hp核酸疫苗pIREShpaA,并经Western印迹法检测到特异性蛋白条带。结论构建了hpaA基因的Hp核酸疫苗,为进一步探索其免疫作用奠定了基础。
Objective To construct a nucleic acid vaccine containing hpaA gene of Helicobacter pylori (Hp). Methods The genomic DNA of Hp standard strain CCUG17874 was extracted. The hpaA gene was amplified from genomic DNA by polymerase chain reaction (PCR) and cloned into pUCmT vector. The sequence of hpaA gene was detected. After a series of digestion and ligation reactions, The eukaryotic expression vector pIRES was transformed into E. coli and the positive clones were screened and identified by PCR and restriction enzyme digestion. The constructed recombinant vector pIREShpaA was transfected into COS7 cells by liposome method. The immunogenicity of pIREShpaA-expressing HpaA protein was detected by SDSPAGE and Western blot. Results The hpaA gene of about 750bp was successfully amplified. The sequencing results showed that the amplified hpaA gene was consistent with the HpapaA sequence. The PCR and restriction enzyme digestion confirmed that the hpaA gene was cloned into the eukaryotic expression vector pIRES and the hpaA gene Hp DNA vaccine pIREShpaA, and detected by Western blot specific protein bands. Conclusion The Hpa DNA vaccine of hpaA gene was constructed, which laid the foundation for further exploring its immunological function.