牛结核分枝杆菌ESAT6-CFP10-BFH融合蛋白原核表达及皮试反应  

Prokaryotic expression of fusion protein ESAT6-CFP10-BFH of mycobacterium bovis tuberculosis and skin test reaction

作　　者：鲍佳鹏 孙悦 张建军 苏岳乐 毕力格 郭宇 巴依尔塔 赵东辉 陈彪 冯紫佳 郑可 秦建华[1] BAO Jiapeng;SUN Yue;ZHANG Jianjun;SU Yuele;BI Lige;GUO Yu;BAYI Erta;ZHAO Donghui;CHEN Biao;FENG Zijia;ZHENG Ke;QIN Jianhua(Hebei Agricultural University,Baoding,Hebei 100086,China;Shijiazhuang Shengbo Biotechnology Co.,Ltd,Shijiazhuang 050031,China;Wulanchabu Animal Disease Prevention and Control Center,Wulancha-bu,Inner Mogolia 012000,China;Inner Mongolia Autonomous Region Animal Disease Preven-tion and Control Center,Huhehaote,Inner Mongolia 010010,China;Inner Mongolia Tongliao Animal Disease Prevention and Control Center,Tongliao,Inner Mongolia 028000,China;Inner Mongolia Tongliao Animal Quarantine Technology Service Center,Tongliao,Inner Mongolia 028001,China;Hebei University of Science and Technology,Shijiazhuang 050018,China;People's Hospital of Guangxi Zhuang Autonomous Region,Nanning 530021,China)

机构地区：[1]河北农业大学,河北保定100086 [2]石家庄圣博生物科技有限公司,河北石家庄050031 [3]乌兰察布市动物疫病预防控制中心,内蒙古乌兰察布012000 [4]内蒙古自治区动物疫病预防控制中心,内蒙古呼和浩特010010 [5]内蒙古通辽市动物疫病预防控制中心,内蒙古通辽028000 [6]内蒙古通辽市动物检疫技术服务中心,内蒙古通辽028001 [7]河北科技大学,河北石家庄050018 [8]广西壮族自治区人民医院,广西南宁530021

出　　处：《中国兽医学报》2023年第12期2502-2508,共7页Chinese Journal of Veterinary Science

基　　金：石家庄圣博生物科技有限公司自筹资助项目。

摘　　要：为了改善ESAT6-CFP10融合蛋白检测牛结核分枝杆菌感染存在灵敏度低的问题,应用铁蛋白骨架显著性提升EC(ESAT6-CFP10)法的灵敏度,将ESAT6、CFP10和牛源铁蛋白重链(BFH)用柔性多肽连接,按照ESAT6-CFP10-BFH的串联组合方案设计基因序列,构建pET-28a-EC-BFH重组质粒,转化至大肠杆菌BL21(DE3)感受态细胞中。使用自诱导培养基对铁蛋白EC融合抗原(EC-BFH蛋白)进行诱导表达,亲和层析镍柱法纯化EC-BFH蛋白,使用蛋白印迹(Western blot)对EC-BFH蛋白进行鉴定及纯度测定。将EC-BFH蛋白对健康豚鼠进行皮试,通过观察皮试部位是否会引起炎性反应,验证EC-BFH蛋白的特异性。对牛结核分枝杆菌致敏成功的豚鼠,以提纯牛型结核菌素(PPDB)作为对照,检测EC蛋白和EC-BFH蛋白效价对比阳性率。结果表明:EC-BFH蛋白的相对分子质量约为48.4 kDa,与预期相同,说明成功纯化EC-BFH蛋白。EC-BFH蛋白对健康豚鼠进行皮试24 h后无炎症反应,说明EC-BFH蛋白特异性良好。根据阳性率数据显示EC-BFH蛋白效价远远高于EC蛋白;在注射量为1,10和20μg时,EC-BFH蛋白皮试的阳性率均高于EC蛋白,尤其注射量在1μg时,EC蛋白和EC-BFH蛋白的阳性率分别为16.6%和100%,且EC-BFH蛋白与PPDB的阳性率接近。结果表明EC-BFH融合蛋白提高了EC法检测牛结核分枝杆菌感染的灵敏度,增强了皮试反应效果,作为检测牛结核分枝杆菌感染的诊断试剂有很大的研究价值。To improve the sensitivity of the ESAT6-CFP10 fusion protein in detecting Mycobacterium tuberculosis infection,the sensitivity of the EC(ESAT6-CFP10)method was significantly improved by using the ferritin backbone.ESAT6,CFP10 and bovine ferritin heavy chain(BFH)were ligated with flexible peptides,and the gene sequence was designed according to the tandem combination scheme of ESAT6-CFP10-BFH to construct the pET-28a-EC-BFH recombinant plasmid,which was transformed into E.coli BL21(DE3)competent cells.Ferritin EC fusion antigen(EC-BFH protein)was induced by self-induction medium,EC-BFH protein was purified by nickel column affinity chromatography,and EC-BFH protein was identified,and its purity was determined by Western blot.EC-BFH protein was tested in healthy guinea pigs to verify the specificity of EC-BFH protein by observing whether the skin test site would cause an inflammatory response.In guinea pigs sensitized with Mycobacterium bovis,purified protein derivative of bovine tuberculin(PPDB)was used as a control to determine the positive rate of EC protein and EC-BFH protein titer.The results showed that the relative molecular mass of EC-BFH protein was about 48.4 kDa,which was the expected value,indicating that EC-BFH protein was successfully purified.The EC-BFH protein showed no inflammatory reaction after a 24-hour skin test in healthy guinea pigs,indicating that the EC-BFH protein had good specificity.According to the positive rate data,the titer of EC-BFH protein was much higher than that of EC protein,and the positive rates of EC-BFH protein skin test were higher than those of EC protein at injections of 1,10,20μg,especially at injections of 1μg,the positive rates of EC protein and EC-BFH protein were 16.6% and 100%,respectively,and the positive rates of EC-BFH protein and PPDB were close to each other.These results suggest that the EC-BFH fusion protein improves the sensitivity of EC method for the detection of Mycobacterium bovis infection and enhances the skin test reaction effect,which has great re

关 键 词：ESAT6-CFP10融合蛋白 牛结核分枝杆菌 提纯牛型结核菌素 

分 类 号：S852.61[农业科学—基础兽医学] R535[农业科学—兽医学]