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研究羊布鲁菌外膜蛋白omp2b基因的克隆,原核表达,以及纯化。根据羊布鲁菌M5株外膜蛋白omp2b蛋白基因序列设计引物,扩增出大小约为1 130 bp的目的基因片断,克隆入融合表达载体pGEX-4T-1,构建重组质粒pGEX-4T-1-omp2b。在大肠埃希菌中将该蛋白表达并用亲和层析法纯化。用Western-blot分析方法鉴定纯化蛋白。结果成功构建了pGEX-4T-1-omp2b原核表达载体并在大肠埃希菌中表达了omp2b基因,纯化后所获得的蛋白与目的蛋白大小一致且与兔抗布鲁菌血清发生特异性反应,说明其为布鲁菌omp2b蛋白。本研究成功构建了pGEX-4T-1-omp2b原核表达载体,并且在大肠埃希菌中进行表达,纯化的omp2b蛋白具有良好的免疫原性。
To study the Cloning, prokaryotic expression and purification of the omp2b gene of goat brucella outer membrane. According to the sequence of omp2b protein gene of M. bassiana M5 strain, a fragment of about 1 130 bp was amplified and cloned into the fusion expression vector pGEX-4T-1 to construct the recombinant plasmid pGEX-4T-1 -omp2b. The protein is expressed in Escherichia coli and purified by affinity chromatography. The purified protein was identified by Western-blot analysis. Results The prokaryotic expression vector pGEX-4T-1-omp2b was successfully constructed and the omp2b gene was expressed in Escherichia coli. The purified protein was expressed in the same size as the target protein and reacted specifically with rabbit anti-Brucella serum. It is Brucella omp2b protein. In this study, the prokaryotic expression vector pGEX-4T-1-omp2b was successfully constructed and expressed in Escherichia coli. The purified omp2b protein has good immunogenicity.