牛分枝杆菌esat6和cfp10蛋白的表达及在临床检测中的应用  

Expression and Clinical Significance of Mycobacterium bovis esat6 and cfp10 Protein

作　　者：解晓莉 韩猛 刘来兴[1] 王基隆 杨美 张云飞 丁家波[2] 张亮[1] 杨宏军[1] 

机构地区：[1]山东省农业科学院奶牛研究中心,山东济南250000 [2]中国兽医药品监察所,北京100081

出　　处：《山东农业科学》2017年第11期120-123,共4页Shandong Agricultural Sciences

基　　金："十三五"国家重点研发计划项目"牛羊重要疫病诊断与检测新技术研究"(2016YFD0500900);现代农业(奶牛)产业技术体系科学家岗位项目(CARS-36);山东省自然科学基金三院联合项目"牛型分枝杆菌ESAT6和CFP10调控巨噬细胞炎性反应的功能研究"(ZR2015YL069);山东省农业科学院农业科技创新工程项目"主要畜禽疫病监测和预警"(CXGC2016A10)

摘　　要：本研究以牛分枝杆菌H37Rv基因组DNA为模板,扩增esat6和cfp10基因,构建重组表达载体p ET-32a(+)-esat6和p ET-32a(+)-cfp10,并通过原核表达系统分别表达重组esat6和cfp10蛋白,经Ni-NTA亲和层析法纯化后,对目的蛋白进行SDS-PAGE和Western blot验证,BCA法测定目的蛋白浓度,最后将east6和cfp10蛋白混合后用于牛结核病临床检测,并对检测数据进行分析。结果表明,牛分枝杆菌esat6和cfp10蛋白在大肠杆菌系统中获得高效表达;其混合蛋白应用于结核病检测具有较高的特异性,且在皮肤变态反应和IFN-γ释放试验两种检测方法中具有较高的符合率。说明相较于提纯结核菌素(PPD),esat6和cfp10混合蛋白具有较高的特异性和稳定性,将有希望替代PPD用于牛结核病检测。In this study,the esat6 and cfp10 gene were cloned with genomic DNA of Mycobacterium bovis strain H37Rv as template. Recombinant expression vectors p ET-32 a( +)-esat6 and p ET-32 a( +)-cfp10 were constructed to express esat6 and cfp10 protein through the prokaryotic expression system. The recombinant proteins were purified by Ni-NTA affinity chromatography and verified with SDS-PAGE and Western blot. Furthermore,the protein concentrations were determined with BCA method. Then the two proteins were mixed to detect the BTB( bovine tuberculosis) and the results were analyzed. The results showed that esat6 and cfp10 protein were highly expressed in E. coli. There were high specificity and coincidence rate in skin allergic reaction and IFN-γ release test using mixed protein as stimulator. This study confirmed that compared with PPD,esat6 and cfp10 mixed protein had higher specificity and stability and was a promising alternative in BTB detection.

关 键 词：牛分枝杆菌 esat6和cfp10蛋白 皮肤变态反应 IFN-γ释放试验 

分 类 号：S858.23[农业科学—临床兽医学]