结核分枝杆菌pdhA基因的原核表达及其免疫原性分析  

Prokaryotic expression of pdhA gene from Mycobacterium tuberculosis and immunogenicity analysis of the expressed product

作　　者：王华南[1] 亓英芳[1] 朱婷[1] 于申业[1] 刘慧芳[1] 司微[1] 杨盛[2] 王加明[2] 冯拥军[2] 李素兰[2] 于秀婷[1] 王春来[1] 刘思国[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室动物细菌病研究室,黑龙江哈尔滨150001 [2]新疆生产建设兵团农十师畜牧兽医站,新疆阿勒泰836000

出　　处：《中国兽医科学》2011年第7期697-700,共4页Chinese Veterinary Science

基　　金：国家重点基础研究发展计划(973)项目(2006CB504400);国家科技基础性工作专项(2008FY210200);中央级公益性科研院所基本科研业务费专项(ZGKJ201015)

摘　　要：以结核分枝杆菌H37Rv菌株的基因组DNA为模板,采用PCR方法扩增出1 100bp的pdhA(pyruvate dehydrogenase E1component alpha subunit)基因,经限制性酶切后,与pET-28a载体连接,转化到大肠杆菌BL21中,重组菌经1mmol/L IPTG诱导表达了pdhA蛋白,并用Ni-His-resin纯化了目的蛋白,经Western-blot分析鉴定了目的蛋白的免疫原性。结果显示,重组质粒的双酶切鉴定和DNA测序均正确;SDS-PAGE和Western-blot分析结果显示,在44ku处获得一目的条带,且具有很好的免疫原性。本研究成功获得了高纯度的重组蛋白pdhA,为深入研究其功能奠定了基础。The pdhA（pyruvate dehydrogenase E1 component alpha subunit） gene was amplified from the genome of Mycobacterium tuberculosis H37Rv strain by PCR,cloned into pET-28a vector,and then transformed into Escherichia coli BL21.The recombinant plasmid was confirmed by restriction digestion and DNA sequencing.The pdhA protein was expressed in resultant strains induced with 1 mmol/L IPTG.The recombinant protein was purified with Ni-His-resin and the immunogenicity of it was identified by Western-blot.The recombinant protein with good immunogenicity was confirmed to be about 44 ku in size by SDS-PAGE.The results showed that the recombinant expression vector pET28a-pdhA was successfully constructed and the expressed recombinant protein with high purity provided the foundations for basis of further studies on the function of pdhA protein.

关 键 词：结核分枝杆菌 pdhA基因 原核表达 

分 类 号：S852.618[农业科学—基础兽医学]