幽门螺杆菌融合蛋白HspA-UreB的表达和免疫学活性

Expression and immunocompetence of HspA-UreB fusion protein of Helicobacter pylori

目的:构建表达幽门螺杆菌(H pylori)融合蛋白HspA-UreB 的重组表达质粒,并研究其免疫学活性. 方法:定向克隆方法将郑州分离Hp菌株MEL-HP27的hspA 和ureB基因融合连接入原核表达载体pET30(a),构建重组表达质粒pET-HU27.该质粒转化大肠杆菌BL21后经IPTG诱导,SDS-PAGE分析融合蛋白HspA-UreB表达情况.Ni2+柱亲和层析纯化融合蛋白,与小鼠免疫血清进行Western blot分析,检测融合蛋白的免疫反应性. 结果:特异PCR法与质粒酶切鉴定证实重组表达质粒pET- HU27构建成功.SDS-PAGE分析显示在82.1 KDa处出现特异蛋白带,占菌体总蛋白的21%,亲和层析法获得纯度为91%的纯化融合蛋白,经免疫小鼠制备的血清可以识别该融合蛋白. 结论:成功构建表达H pylori HspA-UreB融合蛋白的重组表达质粒,表达的融合蛋白具有良好的免疫反应性.

AIM: To construct recombinant expression vector expressing HspA-UreB fusion protein of Helicobacter pylori(H pylori), and to determine its immunoreactivity, in order to develop gene recombinant vaccine against H pylori infection. METHODS: The hspA and ureB genes were amplified by PCR from H pylori MEL-HP27 isolated in Zhengzhou and cloned directionally into vector pNEB193. These two genes were restricted by using two corresponding restriction enzyme separately and cloned together into the fusion expression vector pET-30 (a), and the recombinant plasmid was then used to transform E.coli BL21 (DE3). The positive clones were identified by PCR and restriction enzyme digestion. The recombinant fusion protein HspA-UreB was induced to express from E.coli by IPTG and was analyzed by SDS-PAGE. The fusion protein was purified by use of Ni2+ affinity chromatography and then used to immunize mice. The immunogenecity and immunoreactivity of the fusion protein were analyzed by Western blot. RESULTS: The hspA-ureB fusion gene was amplified from the recombinant fusion expression plasmid pET-HU27 (pET-HspA-UreB) by PCR, and also the hspA-ureB fusion gene fragment was produced from these plasmids after restriction enzyme digestion. SDS-PAGE and optical density scanning indicated that the fusion protein was expressed in the re-combinant vaccine strain BL21 (pET-HU27) as a protein with 82.1 KDa of molecular weight that accounted for 21% of the total bacterial protein. The purity of fusion protein was 91%. Western blot analysis of the purified fusion protein confirmed that it could specifically be recognized by mouse serum. CONCLUSION: A recombinant vaccine candidate strain expression fusion protein HspA-UreB of H pylori is constructed and identified successfully, and purified fusion protein has strong immunoreactivity.