人幽门螺杆菌26000外膜蛋白与热休克蛋白双价疫苗的构建和表达及抗原性研究  

Construction, expression and antigenic study of bivalent vaccine candidate with 26 000 OMP and heat short protein A of human Helicobactor pylori

作　　者：姜政[1] 蒲丹 黄爱龙[2] 陶小红[1] 王丕龙[1] 

机构地区：[1]重庆医科大学第一附属医院消化科,400016 [2]重庆医科大学肝炎研究所,400016

出　　处：《中华医学杂志》2003年第10期862-867,共6页National Medical Journal of China

摘　　要：目的　构建含人幽门螺杆菌 (Hp)热休克蛋白A(HspA)和 2 6 0 0 0外膜蛋白 (OMP)编码基因的重组载体 ,进行核苷酸序列分析 ,并在大肠杆菌BL2 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编码基因片段 ,将目的基因HspA与载体 pET32a(+)经kpnⅠ、BamHI双酶切后 ,进行连接、测序 ;同时将重组载体 pET32a(+) /HspA和 pET32a(+) /Omp2 6分别经HindⅢ、BamHI双酶切 ,通过凝胶电泳回收 pET32a(+) /HspA和 2 6 0 0 0OMPDNA片段 ,经T4连接酶将HspA和 2 6 0 0 0OMP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(DE30 )中表达 ,表达产物经纯化后 ,以western印迹法检测其抗原性。结果　经酶切、测序分析表明 ,插入的基因片段为HpHspA和2 6 0 0 0外膜蛋白编码基因 ,由 95 1bp组成 ,与GenBank报道的相比较 ,有 1.15 %的bp发生变异 ,1 2 6 %的氨基酸残基改变 ;经SDS PAGE分析发现 ,融合基因表达的蛋白相对分子质量 (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 19.96 % ;重组蛋白经含Ni2 + 琼脂糖树脂纯化后 ,其纯度达 95 %以上 ;western印迹法检测显示 ,该重组蛋白?Objective To construct a recombinant vector containing gene encoding heat shock protein A(HspA) and outer membrane protein(OMP) with relative molecule mass ( Mr ) of 13 000 and 26 000 respectively from human Helicobacter pylori (Hp) and be expressed in E. coli BL21, as well as analyse its antigenic for the exploiting vaccine of Hp. Methods The target gene encoding heat shock protein A was amplified from Hp chromosome by PCR. And then digested by restricted endonuclease enzyme of kpnⅠ, Bam H I simultanously, and inserted into the prokaryotic expression vector pET32a(+)digested by corresponding restricted endonuclease enzyme. The recombinant vector was used to select and transform for sequence analysis. After pET32a(+)/HspA and pET32a(+)/Omp 26 digested by restricted endonuclease enzyme of Hind Ⅲ, Bam H I simultanously, the pET32a(+)/HspA and 26 000 OMP were taken out of agarose electrophoresis, and connected by T4 ligase. The recombinant vector pET32a(+)/HspA-Omp 26 was used to select and transform, meanwhile expressed in E. coli BL21(DE3). The antigenic of recombinant fusion protein was analysed by western blotting. Results Enzyme digestion analysis and sequencing showed that the target genes was found to be 951 base pairs, and had been inserted into recombinant vector, but as compared with gene reported by GenBank, 1.15 % of the gene mutation and 1.26 % 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 of expressed product was 59×103, while Mr of protein expressed by pET32a(+)of them was about 20×103, and soluble expression product accounted for 19.96 % of total bacterial protein. After purification with Ni-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 and monocloned antibody of 26 000 OMP, suggesting th

关 键 词：幽门螺杆菌 热休克蛋白质类 细菌外膜蛋白质类 主动免疫疗法 

分 类 号：R392.1[医药卫生—免疫学]