结核分枝杆菌ESAT-6重组二聚体的表达、纯化及其在血清学诊断中的初步应用  

Expression and Purification of Recombinant Dimmer ESAT-6 of Mycobacterium tuberculosis and Its Preliminary Application in Serological Diagnosis of Tuberculosis

作　　者：郭建[1,2,3] 范小勇[2,4] 马辉 卢水华[2] 熊延青[2] 朱友林[1] 李忠明 郭盛淇[4] 

机构地区：[1]南昌大学生命科学学院,南昌330031 [2]复旦大学附属上海市公共卫生临床中心,上海201508 [3]上海海规生物科技有限公司,上海201203 [4]上海生物制品研究所,上海200052

出　　处：《中国生物制品学杂志》2009年第2期128-131,共4页Chinese Journal of Biologicals

基　　金：上海市卫生局级课题(2008057)

摘　　要：目的表达和纯化结核分枝杆菌早期分泌蛋白ESAT-6重组二聚体(rdESAT-6),并探讨其在结核病血清学诊断中的价值。方法以HG856A核酸疫苗质粒为模板,经PCR扩增获得2×esat-6基因,克隆至pET-28a质粒,构建原核表达质粒pET2E6;转化大肠杆菌BL21(DE3),IPTG诱导表达;用Ni2+柱亲和层析纯化重组蛋白,并用Western blot鉴定其反应原性,ELISA检测其敏感性和特异性。结果ESAT-6重组二聚体以包涵体形式表达,表达量约占菌体总蛋白的35%;纯化的rdESAT-6纯度可达95%,可与ESAT-6小鼠抗血清发生特异性反应;用于结核病血清学诊断的敏感性为30%,特异性为95.8%。结论已成功表达并纯化了rdESAT-6,可作为结核病血清学诊断或皮试检测用的抗原之一。Objective To express and purify the ESAT-6 dimmer of Mycobacterium tuberculosis and investigate its preliminary application in the serological diagnosis of tuberculosis. Methods 2xeast-6 gene was amplified by PCR using DNA vaccine HG856A as template and cloned into plasmid pET-28a. The constructed recombinant vector pET2E6 was transformed to E. coli BI21 （DE3） for expression under induction of IPTG. The target protein was purified by nickel ion affinity chromatography, identified for reactogenicity by Western blot, and evaluated for sensitivity and specificity by ELISA. Results Recombinant dimmer ESAT-6 （rdE- SAT-6） was expressed in a form of inclusion body, up to 35% of total lysates. The expressed product reached a purity of 95% after purification and reacted specifically with murine antisera against ESAT-6. The sensitivity and specificity of purified rdESAT-6 used for serological diagnosis of tuberculosis were 30% and 95.8% respectively. Conclusion Recombinant ESAT-6 dimmer was success- fully expressed and purified, which might be used as one of the candidate antigens for serological diagnosis or skin test of tuberculosis.

关 键 词：结核分枝杆菌 ESAT-6重组二聚体 表达 纯化 血清学诊断 

分 类 号：R378.911[医药卫生—病原生物学] Q789[医药卫生—基础医学]