幽门螺杆菌hpaA核酸疫苗的构建及免疫原性检测

Construction of Helicobacter pylori hpaA nucleic acid vaccine and detection of its immunogenicity

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 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 DH5α, 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.