融合蛋白Rv3872/CFP-10/ESAT-6的制备及在牛结核诊断中的初步应用  

Preparation of Fusion Protein Rv3872/CFP-10/ESAT-6 and Primary Application in Detection of Bovine Tuberculosis

作　　者：刘冬光[1,2] 廖娟红[1,2] 于清龙[1,2] 陈颖钰[1,2] 陈焕春[1,2] 郭爱珍[1,2] 

机构地区：[1]华中农业大学农业微生物学国家重点实验室,武汉430070 [2]华中农业大学动物医学院,武汉430070

出　　处：《中国奶牛》2009年第10期7-11,共5页China Dairy Cattle

基　　金："973"计划重要传染病基础研究专项(2006CB504401);国家"十一五"科技支撑计划项目(编目编号2006BAD04A05);农业公益性行业科研专项(200803018)

摘　　要：本研究通过融合表达Rv3872、CFP-10和ESAT-6蛋白,以改良牛结核病特异性鉴别诊断抗原CFP-10-ESAT-6,提高牛结核抗体鉴别诊断法的灵敏度。利用PCR方法,克隆牛分枝杆菌RD1区上述三个基因,通过酶切连接方法,将三个基因串联在pET-28a载体上,基因之间用Linker序列连接。融合基因在大肠杆菌内经IPTG0.4mmol/L诱导3h,目的蛋白经SDS-PAGE证实表达在上清中,Western-blot分析证实表达蛋白具有免疫原性。以所表达的融合蛋白作为包被抗原建立间接ELISA,检测了44份牛分枝杆菌感染背景清楚的临床奶牛血清。26份阳性血清中,Rv3872-CFP-10-ESAT-6融合蛋白ELISA检出阳性样本18份,检测灵敏度为69%,而CFP-10-ESAT-6融合蛋白ELISA只检出阳性样本4份。18份阴性血清中,两种ELISA均检出17份阴性血清,特异性都为94%(17/18)。因此,Rv3872-CFP-10-ESAT-6融合蛋白在对牛结核抗体检测特异性没有降低的情况下,敏感性获得了显著提高,这对牛结核病的早期鉴别诊断方法的建立具有重要意义。In order to improve the effect of the bovine M. boris specific antigen CFP-10-ESAT-6 in detection of M. bovis antibody, a fusion protein of Rv3872, CFP-10 and ESAT-6 was expressed and purified. Three genes in M. bovis RD1 region were separately amplified by PCR, linked with a linker sequence and cloned into pET-28a expression vector. The recombinant plasmid was transformed into E.coli BL21 （DE3） and the fusion protein was expressed after induction of 3h with 0.4mmol/L IPTG. The fusion protein was confirmed with SDS-PAGE. The Western-blot analysis demonstrated that the fusion protein has immunogenieity. The indirect ELISA was developed with this fusion protein and utilized for detection of 44 cow serum samples including 26 positive and 18 negative sera. Among 26 positive sera, 18 positive sera were detected to be positive with the sensitivity of 69% when the fusion protein Rv3872-CFP-10-ESAT-6 based indirect ELISA was used.However, the fusion protein CFP-10-ESAT-6 based indirect ELISA only detected 4 samples. On the other hand, both the Rv3872-CFP-10- ESAT-6 and CFP-10-ESAT-6 based indirect ELISA detected 17 among 18 negative sera with the specificity of 94%（17/18）. It suggests that Rv3872-CFP-10-ESAT-6 fusion protein has a higher sensitivity than CFP-10-ESAT-6 in detection of M. bovis antibody.

关 键 词：牛结核 Rv3872 CFP-10 ESAT-6 诊断 

分 类 号：S858.23[农业科学—临床兽医学] Q78[农业科学—兽医学]