结核分枝杆菌csdA基因的原核表达及其免疫原性  

Prokaryotic expression and identification of csdA gene from Mycobacterium tuberculosis

作　　者：王华南[1] 亓英芳[1] 杜艳芬[1] 刘慧芳[1] 司微[1] 于申业[1] 王加明[2] 杨盛[2] 李素兰[2] 冯拥军[2] 倪洪波[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》2010年第12期1232-1235,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项目(2008FY210200)

摘　　要：以结核分枝杆菌H37Rv基因组DNA为模板,采用PCR方法扩增出1 690 bp的csdA(cold-shock dead-box protein A)基因,经限制性酶切后,与pET28a载体连接,转化到大肠杆菌BL21中后,重组菌经0.2 mmol/L IPTG诱导表达了csdA蛋白及Ni-His-resin纯化蛋白,经Western-blot分析,鉴定了目的蛋白的反应原性。试验结果表明,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64 ku处获得一目的条带,具有很好的免疫原性。证实,成功构建了重组表达质粒pET28a-csdA,获得了高纯度的重组蛋白,为深入研究csdA蛋白的功能奠定了基础。The csdA（cold-shock dead-box protein A） gene was amplified by PCR from the genome of Mycobacterium tuberculosis H37Rv strain,and then it was cloned into pET28a vector.The product of ligation was transformed into Escherichia coli BL21.The recombinant plasmid was confirmed by restriction digestion and DNA sequencing.The CsdA protein was expressed inducing with 0.02mmol/L IPTG.The recombinant protein was purified with Ni-resin and the immunogenicity of the recombinant protein was identified by Western-blot.The recombinant protein was confirmed to be about 64ku in size.The results showed that the recombinant expression plasmid pET28a-csdA was successfully constructed and the recombinant protein with high purity laid the foundation for further studies on the csdA protein.

关 键 词：结核分枝杆菌 csdA基因 原核表达 

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