幽门螺杆菌Tipα重组蛋白的表达、纯化及鉴定

Construction,expression and purification of recombinant H.polyri Tip-α protein

目的:克隆幽门螺杆菌(H.polyri,HP)的Tipα基因,构建含有Tipα的重组载体,在大肠杆菌表达并纯化以获得Tipα蛋白。方法:从HP的26695菌株扩增位于HP0596的Tipα基因,将Tipα基因与pET28a载体(His标签)同时经BamHI和XhoI酶切、纯化及连接后,构建含有Tipα的重组载体pET28a-Tipα,重组载体转化至大肠杆菌并表达,表达产物以Ni-NTA亲和层析纯化,Western blot进一步鉴定。结果:克隆Tipα基因,经测序证实与Genbank对比,二者同源性为100%。大肠杆菌表达产物,纯化后SDS-PAGE显示相对分子量为23 ku。Western blot证实纯化产物与抗Tipα抗体特异性结合。结论:成功克隆并表达出HP的Tipα蛋白,为进一步研究Tipα的功能奠定基础。

Objective：To construct a recombinant vector containing Tip-α of H.polyri,and transfect it in E.coli to purify recombinant protein.Methods：By PCR technique,Tip-α cDNA was amplified,and then cloned into the pET28a vector.The recombinant plasmid was transformed into E.coli.The correct clone was identified by sequence analysis.The expression product was purified by nick-nitrilotriacetic acid（Ni-NTA） metal-affinity chromatography and analyzed by SDS-PAGE and Western blot methods.Results： Enzyme digestion analysis and sequencing assay showed that Tip-α gene had been successfully inserted into the vector.SDS-PAGE showed a 23 ku protein identified by Western blot analysis.Conclusion：A recombinant plasmid containing Tip-α has been constructed and expressed successfully.