牛分支杆菌MPT83基因的表达及重组蛋白的纯化  

Expression of MPT83 gene from Mycobacterium bovis and purification of its recombinant protein

作　　者：鲁俊鹏[1] 罗满林[1] 宋延华 邹潍力[1] 

机构地区：[1]华南农业大学 兽医学院,广东广州510642 [2]广东温氏食品集团有限公司,广东新兴527439

出　　处：《中国兽医科学》2006年第5期366-370,共5页Chinese Veterinary Science

基　　金：广东省"十五"重大科技攻关计划项目(2003A20403)

摘　　要：从牛分支杆菌培养物中抽提总DNA,扩增了MPT83基因,并克隆入pMD 18-T Simple Vector,将测序正确的目的基因插入表达载体pET-32a(+)中,转化BL21(DE3)宿主菌,IPTG诱导表达,用亲和层析方法对表达蛋白进行了纯化。经测序,构建的克隆质粒pMD-MPT83与Gen- Bank中的序列一致;构建的表达质粒pET-MPT83经PCR和BamHⅠ+HindⅢ双酶切鉴定构建正确;经SDS-PAGE分析,在约42 ku处出现新的蛋白条带,4 h后表达量即达到高峰;Western- blotting分析表明,融合蛋白能够被牛分支杆菌阳性血清所识别;纯化的表达蛋白经SDS-PAGE电泳,出现清晰的单一条带。表明MPT83基因原核表达载体构建成功。The total DNA was extracted from the culture of Mycobacterium bovis. MPT83 gene fragment was amplified by PCR, the PCR product was cloned into pMD 18 T Simple Vector and then se quenced. The confirmed MPT83 gene was cloned into plasmid pET-32a （＋） and then transformed into BL21（DE3） where it was induced to express by IPTG. The expressed protein was purified through affinity chromatography. Sequencing results revealed that target sequence was identical to that of the gene available in GenBank. The expression vector pET-32a（＋） was constructed and verified by PCR amplification and digestion with endonucleases BamH Ⅰand Hind Ⅲ . After induction with IPTG, there was a specific protein band of approximately 42 ku on SDSPAGE gel. Cells induced for 4 hours by IPTG were harvested. Results of Western-blotting analysis indicated that the recombinant protein could react with polyclonal antibody against M. boris. After sonication and affinity chromatography, SDS-PAGE showed only one specific band. These results demonstrated that the MPT83 gene was successfully cloned, its prokaryotic expression vector successfully constructed and purified recombinant protein was obtained through affinity chromatography.

关 键 词：牛分支杆菌 MPT83基因 克隆 原核表达 蛋白纯化 

分 类 号：S852.618[农业科学—基础兽医学] Q786[农业科学—兽医学]