副结核分枝杆菌MAP0862-1595融合蛋白的表达及间接ELISA检测方法的建立  

Expression of MAP0862-1595 Recombinant Protein of Mycobacterium avium subsp.paratuberculosis and Establishment of Indirect ELISA Detection Method

作　　者：李晓宇 马聿田 李阳 梁雯 阿丽雅 吉林台 马慧君 希尼尼根[1] LI Xiaoyu;MA Yutian;LI Yang;LIANG Wen;Aliya;Jilintai;MA Huijun;Xininigen(College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot 010011,China;Alxa East County Bayanhot Town Comprehensive Support and Technology Promotion Center,Bayanhot 750300,China)

机构地区：[1]内蒙古农业大学兽医学院,呼和浩特010011 [2]阿拉善左旗巴彦浩特镇综合保障和技术推广中心,巴彦浩特750300

出　　处：《内蒙古农业大学学报（自然科学版）》2024年第6期1-9,共9页Journal of Inner Mongolia Agricultural University(Natural Science Edition)

基　　金：内蒙古自治区高等学校科学研究项目(NJZZ22490);鄂尔多斯市科技重大专项项目(2022EEDSKJZDZX025)。

摘　　要：为了建立基于重组蛋白MAP0862-1595的山羊副结核病诊断方法,本研究筛选副结核分枝杆菌的2个基因MAP0862与MAP1595,通过重叠延伸PCR技术将获得MAP0862-1595融合基因与重组质粒载体pEASY-T1及表达重组质粒载体pET30a连接,通过Western-blot方法验证MAP0862-1595重组蛋白的反应原性,通过以纯化后的重组蛋白MAP0862-1595作为包被抗原和反应条件优化构建间接ELISA法。结果显示:重叠延伸PCR扩增出MAP0862和MAP1595融合基因片段,长度为1601 bp;成功构建克隆重组质粒pEASY-MAP0862-1595和表达重组质粒pET30a-MAP0862-1595;MAP0862-1595重组蛋白大小约为70 kDa,主要以包涵体形式存在;Western-blot验证该蛋白能与副结核分枝杆菌阳性血清特异性结合反应。将纯化后MAP0862-1595蛋白以1μg/孔包被,一抗血清以1∶400稀释,以5%脱脂乳进行封闭,二抗以1∶50000稀释,成功构建ELISA检测法。ELISA方法具有良好的敏感性、特异性和重复性,为副结核病的检测及试剂盒的开发提供了技术支持。In order to establish a diagnostic method for the goat paratuberculosis based on the recombinant protein MAP0862-1595,this study screened two genes,i.e.,the MAP0862 and MAP1595,from the Mycobacterium avium subsp.paratuberculosis.The fu⁃sion gene MAP0862-1595 was obtained by using the overlapping extension PCR technology,subsequently linked to the recombinant plasmid vector pEASY-T1 and the recombinant plasmid vector pET30a,respectively.The reactogenicity of the recombinant protein of MAP0862-1595 was verified by using the Western-blot method,and the purified recombinant protein MAP0862-1595 was used as a coating antigen to construct an indirect ELISA method through optimization of reaction conditions.The results showed that the over⁃lapping extension PCR successfully amplified the fusion gene fragment of MAP0862 and MAP1595,with a length of 1601 bp.The cloned recombinant plasmid pEASY-MAP0862-1595 and the recombinant plasmid pET30a-MAP0862-1595 were successfully con⁃structed,and the size of the recombinant protein of MAP0862-1595 was about 70 kDa,primarily existing in the form of inclusion bodies.The Western-blot analysis confirmed that this protein specifically bound to the positive sera of Mycobacterium avium subsp.paratuberculosis.The purified MAP0862-1595 protein was coated at a concentration of 1µg/well,with the primary antibody diluted at 1∶400 and blocked with 5%skim milk,while the secondary antibody was diluted at 1∶50000,successfully establishing the ELI⁃SA detection method.The ELISA method demonstrated good sensitivity,specificity,and reproducibility,providing technical sup⁃ports for the detection of paratuberculosis and the development of kits.

关 键 词：副结核分枝杆菌 MAP0862 MAP1595 串联表达 

分 类 号：S855.1[农业科学—临床兽医学]