幽门螺杆菌双亚单位基因疫苗的构建及免疫原性研究

Construction and immunogenicity studies on a fused DNA vaccine with UreB and HspA of Helicobacter pylori

目的:构建幽门螺杆菌(Hp)两个保护性亚单位UreB、HspA融合基因疫苗并观察其免疫原性。方法:从Hp基因组分别扩增出UreB、HspA基因片段,采用重叠延伸PCR方法将两个基因片段构建融合基因,以BglⅡ和XhoⅠ酶切位点克隆入真核表达载体pcDNA3.1(+)中,经测序确认后转染COS-7细胞,Western blot检测目的蛋白表达,胫前肌注射免疫小鼠后收集血清检测特异性抗体效价。结果:经测序后证实成功将融合基因构建到真核表达载体中,免疫印记检测到目的蛋白表达,并具有良好的抗原性,免疫小鼠后能够产生特异性抗体。结论:成功构建Hp双价DNA疫苗,免疫小鼠后显示出良好的免疫原性,为Hp进一步基因疫苗的研究奠定了基础。

To construct a fused H. pylori DNA vaccine consisting of UreB, HpaA gene fragments, and observe its immunogenicity. Methods：The fragment of UreB and HspA genes was obtained by PCR amplification from H. pylori genome DNA, then the fusing gene UH was obtained by SOE PCR（ splicing by overlap extension）. UH was subcloned into an eukaryotic expression vector pcDNA3. 1. Then the fusion protein was identified by Western blot after plasmid pcDNA3. 1-UH was transfected into COS-7 cells and ELISA was used to detect the specific antibody after immunization in mice. Results：The eukaryotic expression plasmid of the fusing gene two fragments was constructed, and a novel fusion protein was expressed after transfection into COS-7 cell. Specific antibody was found in mouse serum after immunization with the DNA vaccine. Conclusion： A fused DNA vaccine of from H. pylori with good immunogenicity has been successfully constructed, which lay a foundation for further investigation on H. pylori DNA vaccine.