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目的构建新型呼肠病毒主要抗原蛋白的编码基因S1的重组原核表达质粒,对其在原核细胞内的表达进行研究。方法将S1基因克隆入原核表达载体pProEX HTa,构建重组原核表达载体pPLS,通过IPTG诱导,利用蛋白电泳和蛋白免疫印迹等方法对其在大肠杆菌中的表达进行研究。结果酶切分析表明重组质粒构建成功。SDS-PAGE和Western-blot的检测结果一致表明,诱导后1~5 h均可检测到目的蛋白的表达,其中以5 h的蛋白表达量最大,且表达的目的蛋白主要以不溶性包涵体的形式存在。结论通过构建重组原核表达质粒,可使目的蛋白σ1得到诱导表达,为后续抗体制备及研究病毒-宿主相互作用提供实验基础。
Objective To construct a recombinant prokaryotic expression plasmid encoding gene S1 of the novel reovirus major antigen protein and study its expression in prokaryotic cells. Methods The S1 gene was cloned into prokaryotic expression vector pProEX HTa to construct recombinant prokaryotic expression vector pPLS. The recombinant plasmid pPLS was induced by IPTG and its expression in E. coli was studied by protein electrophoresis and Western blotting. Results Restriction analysis showed that the recombinant plasmid was successfully constructed. The results of SDS-PAGE and Western-blot showed that the expression of the target protein was detected 1 to 5 h after induction, and the expression of the target protein was the highest at 5 h after induction, and the expressed protein was mainly in the form of insoluble inclusion body exist. Conclusion The expression of the target protein σ1 can be induced by constructing a recombinant prokaryotic expression plasmid, which provides the experimental basis for the subsequent antibody preparation and the study of virus-host interaction.