人幽门螺杆菌VacA与HspA双价疫苗载体的构建

Cloning and onstruction of recombinant vector of bivalent vaccine candidate of human Helicobacter pylori

目的 :构建含人幽门螺杆菌 (Hp)热休克蛋白A(HsPA)和空泡毒素 (VacA)编码基因的重组载体 ,进行核甘酸序列分析 ,为在E .coliBL2 1中表达 ,及其抗原性的研究奠定基础。方法 :利用分子克隆技术 ,扩增HpHspA、VacA编码基因 ,分别将其与载体 pET32a(+)进行酶切、连接 ,同时将筛选的pET32a(+) /HspA和 pET32a(+) /VacA重组载体分别经XhoⅠ、BamHⅠ双酶切 ,通过凝胶电泳回收 pET32a(+) /HspA和VacA片段 ,经T4连接酶将pET32a(+) /HspA和VacA编码基因连接 ,而后转化并筛选含有两种目的基因的重组载体。结果 :经酶切、测序分析表明 ,插入的基因片段为HpHspA和VacA编码基因 ,由10 80bp组成 ,与GenBank报道的相比较 ,有 3.4 %的碱基发生变异 ,1.10 %的氨基酸残基改变 ;但编码的蛋白质特性无本质性改变。结论 :成功地克隆了HpHspA和VacA编码基因 ,并构建了能够同时表达HapA和VacA蛋白的双价疫苗重组载体 ,为Hp蛋白质疫苗和核酸疫苗的研制奠定了良好的基础。

Objective:To construct a recombinant vector containing gene encoding heat shock protein A (HspA) and VacA with Mr 13 000 and 26 000 simultaneously from human Helicobacter pylori(Hp)for study on expression in E.coli BL21,and analysis of their antigenicity.Methods:The target genes encoding HspA and VacaA were amplified from Hp chromosome by PCR,digested by restricted endonuclease enzyme respectively,and inserted into the corresponding enzyme digested prokaryotic expression vector pET32a(+).The recombinant vectors pET32a(+)/HspA and pET32a(+)/VacA were used to select and transform for sequence analysis.After recombinant vectors digested by restricted enzyme of Xhol,BamH Ⅰ simultaneously,the pET32a(+)/HspA and VacA were taken out of agarose electrophoresis,and connected by T4 ligase again.The recombinant vector pET32a(+)/HspA-VacA was used to select and identified by PCR and restricted endonuclease enzyme.Results:Enzyme digestion analysis and sequencing showed that the target genes were found in 1080 base pairs,and had been inserted into recombinant vector,but as compared with gene reported by GenBank,3.40% of the gene mutation and 1.11% of amino acid residues change in Hp happened respectively.Conclusion:The genes coding HspA and VacA with Mr 13 000 and 26 000 respectively are cloned successfully.The results obtained lay the foundation for research on development of Hp protein and DNA vaccine applying to prevention of Hp infection.