人幽门螺杆菌VacA与HspA双价候选疫苗抗原的克隆表达及鉴定

Construction,expression and identification of bivalent vaccine candidate antigens of human Helicobacter pylori

目的 构建含人幽门螺杆菌 (Hp)热休克蛋白A(HspA)和细胞空泡毒素 (VacA)编码基因的重组载体 ,进行核苷酸序列分析 ,并在E coliBL2 1中表达 ,研究其抗原性 ,为疫苗的开发奠定基础。方法 利用分子克隆技术从HpDNA染色体中 ,扩增HspA、VacA编码基因片段 ,将目的基因HspA、VacA与载体 pET32a(+)分别进行双酶切后 ,连接、测序 ;同时将重组载体 pET32a(+) /HspA和 pET32a(+) /VacA分别经Xhol、BamHⅠ双酶切 ,通过凝胶电泳回收 pET32a(+) /HspA和VacADNA片段 ,经T4连接酶将HspA和VacA编码基因通过酶切粘端进行连接 ,而后转化并筛选含有两种目的基因的重组载体 ,并在大肠杆菌BL2 1中表达 ,表达产物经纯化后 ,以Westernblot法检测其抗原性。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 ,本实验克隆的基因有 3 4 0 %的碱基发生变异 ,导致 1 10 %的氨基酸残基改变 ;经SDS -PAGE分析发现 ,融合基因表达的蛋白Mr为 5 9× 10 3 ,其中pET32a(+)表达的蛋白Mr约为 2 0× 10 3 ,可溶性表达产物占全菌总蛋白的 18 96 % ;重组蛋白经Ni2 + -NTA琼脂糖树脂纯化后 ,其纯度达95 %以上 ;用Westernblot法检测显示 ,该重组蛋白可被Hp阳性患者的血清所识别 。

Aim To construct,identify a recombinant vector containing gene encoding heat shock protein A and VacA with Mr 13 000 and 26 000 simultaneously from human Hp.The recombinant vector be expressed in E.coli BL21,as well as analysed its antigenic for the exploiting vaccine of Hp.Methods The target genes encoding HspA and VacaA were amplified from Hp chromosome by PCR.And then digested by restricted endonuclease enzyme respectively,and inserted into the prokaryotic expression vector pET32a(+)digested by corresponding restricted endonuclease enzyme.The recombinant vectors pET32a(+)/HspA and pET32a(+)/VacA were used to select and transform for sequence analysis.After recombinant vector pET32a(+)/HspA and pET32a(+)/VacA digested by restricted endonuclease enzyme of Xhol,BamH Ⅰ simultanously,the pET32a(+)/HspA and VacA were extracted out of agarose electrophoresis,and connected by T4 ligase again.The recombinant vector pET32a(+)/HspA/VacA was used to select and transform,meanwhile expressed in E.coli BL21.The antigenic of recombinant fusion protein was analysed by western blot.Results Enzyme digestion analysis and sequencing showed that the target genes was found to be 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.SDS-PAGE analysis showed that recombinant vector could be expressed in E.coli BL21,its relative molecule mass(Mr)of expressed product was 59×103,while Mr of protein expressed by pET32a(+)was about 20×103,and soluble expression product accounted for 18.96 % of total bacterial protein.After purification with Ni 2+-NTA agarose resion,the purity of recombinant fusion protein was about 95%.The western blot result showed that recombinant fusion protein could be recognized by anti-Hp positive serum,suggesting that this protein had good antigenicity.Conclusion The genes coding HspA and VacA with Mr 13 000 and 26 000 respectively were cloned and expressed successfully.The results obtained lay the foundation for research on development of Hp protein and DNA vaccine and a quickly diagnostic kit applying to detection of Hp infection.