摘要
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 clone,express and purify B.melitensis outer membrane protein omp2b.An 1 130 bp gene was amplified from B.melitensis with PCR method,and then the PCR product was subcloned into vector pGEX-4T-1 and expressed in E.coli.It was purified via affinity chromatography purification system.The purified protein was detected by Western-blot.According to the SDS-PAGE and Western-blot analysis,the recombinant plasmid pGEX-4T-1-omp2b could be expressed successfully in E.coli and the purified protein could react with serum from Brucella infected rabbit.The omp2b gene was constructed and purified successfully and the good immunogenicity of the protein with high efficiency of expression was demonstrated.
出处
《科学技术与工程》
2011年第35期8705-8710,共6页
Science Technology and Engineering
基金
国家自然科学基金(30901281)
陕西省卫生厅2010年科研基金(2010D45)资助
关键词
羊布鲁菌
omp2b蛋白
克隆
表达
纯化
Brucella melitensis omp2b cloning gene expression purification
