牛分枝杆菌CFP-10和ESAT-6基因的融合表达及其抗原活性鉴定  

Fusion expression of CFP-10 and ESAT-6 genes of Mycobacterium bovis and its antigenicity identification

作　　者：柏亚铎[1] 赖平安[1] 史喜菊[1] 高云航[2] 张伟[1] 汪琳[1] 王滨[3] 邓永强[4] 严安[1] 

机构地区：[1]北京出入境检验检疫局,北京100026 [2]吉林农业大学动物科技学院,吉林长春130118 [3]北京市农业局,北京100029 [4]四川省动物疫病预防控制中心,四川成都610041

出　　处：《中国兽医学报》2014年第9期1486-1490,共5页Chinese Journal of Veterinary Science

基　　金：北京市科委资助项目(Z121100001512014)

摘　　要：CFP-10和ESAT-6是牛分枝杆菌的免疫优势抗原,可诱导机体产生IFN-γ,在牛结核病的免疫诊断中发挥重要作用。本试验分别克隆了牛分枝杆菌CFP-10和ESAT-6基因,并将这2个基因融合扩增,构建重组表达质粒pET28a/CFP-10-ESAT-6,重组质粒在大肠杆菌BL21中进行IPTG诱导表达。重组蛋白主要以分泌表达的形式存在于表达上清中,收集上清中的目的蛋白进行Ni亲和层析柱和分子筛两步纯化,蛋白纯度分别达到90%和95%。将纯化的重组蛋白以2mg/L的质量浓度包被酶标反应板,用间接ELISA方法检测不同来源牛血清中的结核病抗体,结果表明,表达的重组融合蛋白具有良好的抗原活性,可有效识别阴、阳性结核病血清。CFP-10 and ESAT-6are the dominant immune antigens of Mycobacterium bovis（M.bovis）,inducing a mount of interferon-γ（IFN-γ）,and plays an important role in immune diagnosis for bovine tuberculosis.CFP-10 and ESAT-6genes were cloned from M.bovis（Vallee Ⅲ）and CFP-10/ESAT-6were amplified as a fusion gene,and recombinant expression plasmid pET28a/CFP-10-ESAT-6was constructed,and expression was induced by IPTG in E.coli BL21.The recombinant proteins are mainly excreted on supernatant,and the target proteins were collected by centrifugation and then purified by Ni 2＋-NTA column chromatography and molecular screen,with the purity of 90% and 95%,respectively.2mg/L purified protein was coated on ELISA plates and known anti-serum of bovine tuberculosis were detected using indirect ELISA,and the results showed that the expressed recombinant protein has good antigenic activities,and can distinguish negative and positive samples without any cross-reaction.

关 键 词：CFP-10 ESAT-6 融合表达 抗原活性 

分 类 号：S852.61[农业科学—基础兽医学]