幽门螺杆菌UreB-Omp11融合蛋白的重组疫苗候选株构建和融合蛋白的表达、纯化及免疫学活性

目的：构建幽门螺杆菌（Hp）UreB-Ompll融合蛋白的重组疫苗候选株，在大肠杆菌中表达UreB-Ompll融合蛋白，并检测其免疫学活性。方法：用PCR方法扩增郑州分离坳菌株MEL-HP27的ureB和ompll基因并用重叠延伸PCR法获得ureB-ompl1融合基因，将融合基因ureB-ompl1插入原核表达载体pET30a（＋）、pET28a（＋）及pMAL-c2X中，筛选出合适的表达系统并进行融合蛋白的表达，采用Westernblot对表达产物进行鉴定，并用Amylose亲和层析法纯化融合蛋白，应用SDS-PAGE方法对纯化产物进行分析，纯化的融合蛋白辅以免疫佐剂皮下免疫小鼠，Westernblot对免疫小鼠血清进行检测。结果：特异PCR法、酶切鉴定并经测序分析后证实融合基因ureB—ompll克隆人表达载体pE330a（＋）、pET28a（＋）与pMAL—c2X中；重组菌TBl（pMAL-ureB—ompl1）经诱导获得了高效表达的MBP-UreB—Ompll融合蛋白，该融合蛋白可以被却免疫小鼠血清和却阳性患者血清中的相应抗体所识别，纯化后的融合蛋白纯度达90％以上。通过大肠杆菌抗原吸收法纯化免疫小鼠血清后，与纯化的融合蛋白进行杂交，结果显示在M，134000处出现特异杂交带，融合蛋白具有良好的免疫原性和免疫反应性。结论：成功地构建并筛选出了却MELHP27融合蛋白UreB-Ompl1的重组疫苗候选株TBl（pMAL-ureB—ompll），为坳蛋白质疫苗和核酸疫苗的研制奠定了基础。

Construction, expression and purification of UreB-Ompll fusion protein of Helicobacter pylori and its immunocompetence

AIM: To construct H.pylori vaccine candidate strain expressing UreB-Omp11 recombinant fusion protein of H.pylori. To express and purify the fusion protein UreB-Omp11 and to determine its immunocompetence. METHODS: The two genes were amplified by PCR, and the fusion gene ureB-omp11 was amplified by over lap extension PCR and then cloned into the fusion expression vector pET30a(+), pET28a(+) and pMAL-c2X. The appropriate expression system was selected, and the recombinant UreB-Omp11 fusion protein was expressed and indentfied by SDS-PAGE and Western blot analysis. Then the fusion protein was purified by MBP affinity chromatography and the purity was indentfied by SDS-PAGE. Then the fusion protein was immunized to mice. The immunized mice sera were analyzed by Western blot with purified fusion protein. RESULTS: The ureB-omp11 fusion gene was correctly insected into pET30a(+) and confirmed by Enzyme digestion and sequencing analysis; Results in SDS-PAGE and optical density scanning demonstrated that this fusion protein MBP-UreB-Omp11 was expressed in the recombinant strain of E.coli TB1(pMAL-ureB-omp11). The fusion protein UreB-Omp11 was recognized by the mice sera immunized by H.pylori, the human sera infected with H.pylori and The purity of fusion protein was 90% after purification. The fusion protein purified could be recognized by corresponding antibody of mice sera immunized by this fusion piotein, This fusion protein has strong immunoantigenicity and immunoreactivity. CONCLUSION: The prokaryotic expression system TB1 (pMAL-c2X-ureB-omp11) was successfully constructed and selected. The results obtained lay the foundation for research on development of protein and DNA vaccine of Hp.