人幽门螺杆菌18000外膜蛋白与HspA双价疫苗的构建和表达  

Construction, expression and antigenicity of bivalent vaccine candidate of human Helicobacter pylori

作　　者：姜政[1] 黄爱龙[2] 蒲丹 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,重庆400016 [2]重庆医科大学肝炎研究所,重庆400010

出　　处：《细胞与分子免疫学杂志》2004年第1期62-66,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目 (No .30 371 31 8)

摘　　要：目的 :构建含人幽门螺杆菌 (Hp)热休克蛋白A(HspA)和Mr为 180 0 0的外膜蛋白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。方法 :用PCR方法从HpDNA染色体中 ,扩增HspA编码基因片段。将目的基因HspA与载体 pET32a(+)分别经kpnⅠ和BamHI双酶切后 ,进行连接、测序。同时将重组载体 pET32a(+) /HspA和pET32a(+) /Omp18,分别经HindIII和BamHI双酶切 ,通过凝胶电泳回收pET32a(+) /HspA和Mr180 0 0OMPDNA片段 ,经T4连接酶将HspA和Mr180 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Ni NTA琼脂糖树脂纯化后 ,以Westernblot分析其抗原性。结果 :经酶切、测序表明 ,插入的基因片段为HpHspA和Mr 为 180 0 0OMP编码基因 ,由 891个碱基组成 ,与GenBank中登录的序列相比较 ,有 1.15 %的碱基发生变异 ,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Mr 为 5 1× 10 3 ,其中 pET32a(+)表达的蛋白Mr 约为 2 0×10 3 ,可溶性表达产物占菌体总蛋白的 18.96 %。重组蛋白经Ni NTA琼脂糖树脂纯化后 ,其纯度达 95 %以上。用Westernblot分析显示 ,该重组蛋白可被Hp阳性患者的血清及抗Mr为 180 0AIM: To construct a recombinant vector containing fused gene of heat shock protein A(HspA) and outer membrane protein(OMP) with M r 18 000, from human Helicobacter pylori (Hp) and express the fusion protein in E.coli BL21. METHODS: The gene encoding HspA was amplified from Hp chromosome by PCR. After digestion with kpnⅠand BamH I, the HspA gene was inserted into the prokaryotic expression vector pET32a(+). After recombinant vectors pET32a(+)/HspA and pET32a(+)/Omp 18 were digested with Hind III and BamH I, the pET32a(+)/HspA and 18 000 OMP gene segments were recovered through agarose electrophoresis, and connected by T4 ligase. The recombinant vector pET32a(+)/HspA-Omp 18 was transformed into E.coli BL21(DE3). The antigenicity of recombinant fusion protein was analysed by Western blot. RESULTS: Enzyme digestion analysis and sequencing showed that the fused gene had 891 base pairs. As compared with gene reported in GenBank, there were 1.15% and 1.26% differences in cloned nucleotide sequence and amino acid sequence respectively. SDS-PAGE analysis showed that recombinant vector could be expressed in E.coli BL21. The relative molecule mass (M r) of expressed fusion protein was 51×10 3. And soluble expression product accounted for 18.96% of total bacterial protein. After purification via Ni-NTA agarose resin, the purity of recombinant fusion protein was about 95%. The Western blot analysis showed that recombinant fusion protein could be recognized by anti-Hp positive serum and anti-18 000 OMP mAb, suggesting that this protein had good antigenicity. CONCLUSION: The fused gene of HspA and OMP is cloned and expressed successfully, which lays the foundation for development of protein and DNA vaccines and a diagnostic kit of Hp infection.

关 键 词：幽门螺杆菌 热休克蛋白A 外膜蛋白 原核表达载体 

分 类 号：R37[医药卫生—病原生物学] Q78[医药卫生—基础医学]