产气荚膜梭菌毒素β1的原核表达及其抗体间接ELISA检测方法的建立  

Prokaryotic expression of Clostridium perfringens Beta1 toxin and development of indirect ELISA method for determination of its antibody

作　　者：马玲玲 马红梅 吴霜 王东 李勇[1,2] 马臣杰 曾瑾[1,2] MA Ling-ling;MA Hong-mei;WU Shuang;WANG Dong;LI Yong;MA Chen-jie;ZENG Jin(Key Laboratory of Ministry of Education for Conservation and Utilization of Special Biological Resources in the Western,Ningxia University,Yinchuan 750021,Ningxia Hui Autonomous Region,China)

机构地区：[1]宁夏大学西部特色生物资源保护与利用教育部重点实验室,宁夏银川750021 [2]宁夏大学生命科学学院,宁夏银川750021

出　　处：《中国生物制品学杂志》2022年第12期1488-1494,共7页Chinese Journal of Biologicals

基　　金：国家自然科学基金(31660719);宁夏回族自治区重点研发计划(东西部合作)(2017BN04);宁夏自然科学基金(2020AAC03073,2021AAC03049)。

摘　　要：目的原核表达产气荚膜梭菌毒素β1(Clostridium perfringens Beta1 toxin,CPB1),建立该毒素抗体的间接ELISA检测方法。方法利用基因工程技术获得cpb1基因,克隆至载体pET32a,构建重组质粒pET32α-cpb1,转化感受态E.coli BL21(DE3),IPTG诱导表达重组蛋白CPB1。以重组蛋白CPB1作为包被抗原,HRP标记的羊抗鼠IgG为二抗,建立CPB1抗体的间接ELISA检测法。并以CPB1的单克隆抗体McAb 1K19为一抗,优化包被蛋白的包被浓度、一抗稀释度、封闭时间及二抗稀释度,确定方法的临界值,验证方法的特异性、精密性、灵敏性。采用建立的方法检测201份正常牛血清样品及阴性、阳性小鼠血清样品。结果表达的重组蛋白CPB1相对分子质量约为54000(CPB1蛋白与硫氧化还原蛋白的融合蛋白),主要以包涵体形式表达。包被蛋白最佳浓度为10μg/mL,一抗最佳稀释度为1∶16384,最佳封闭时间为120 min,二抗最佳稀释度为1∶1000。除B和C型产气荚膜梭菌(Clostridium perfringens,C.p.)外,其他常见食源性致病菌A_(450)均低于0.3;批内及批间精密性CV均<10%;当McAb 1K19稀释度为1∶32768,即浓度为0.002μg/μL时,A_(450)为0.2235,P/N为10.2。201份正常牛血清及阴性小鼠血清均为阴性,阳性小鼠血清为阳性,检测符合率为100%。结论制备的重组蛋白CPB1可用于建立CPB1抗体的间接ELISA检测法,建立的方法具有良好的精密性及较高的灵敏性,可用于大量临床样品的检测。Objective To express Clostridium perfringens Beta1 toxin(CPB1)in prokaryotic cell and develop a indirect ELISA method for determination of its antibody.Methods cpb1 gene was obtained by using genetic engineering technology and cloned to vector pET32a to construct recombinant plasmid which was then transformed to competent E.coli BL21(DE3)and induced by IPTG.Indirect ELISA method for determination of CPB1 antibody was developed using the obtained recombinant protein CPB1 as the coating antigen and goat anti-mouse IgG labeled by HRP as the secondary antibody,which was optimized for the concentration of coating protein,the dilutions of the primary and secondary antibodies and the blocking time,while determined for the critical value and verified for the specificity,precision and sensitivity.The developed method was used to detect 201 serum samples of normal bovines and negative mice as well as two serum samples of positive mice.Results The recombinant protein CPB1 was mainly expressed in the form of inclusion body with a relative molecular mass of 54000(the fusion protein of CPB1 and thioredoxin).The optimal conditions were as follows:coating protein concentration of 10μg/mL,the primary antibody dilution of 1∶16384,blocking time of 120 min and the secondary antibody dilution of1∶1000.Except for Clostridium perfringens(C.p.),of B and C types,A_(450)values of other common foodborne pathogenic bacteria were less than 0.3;CV values of within-run and between-run precision were all less than 10%;When the dilution of McAb 1K19 was 1∶32768,bovines and negative mice were all negative,while those of positive mice were positive,indicating a 100%coincidence rate of detection.Conclusion An indirect ELISA method for determination of CPB1 antibody was developed using the recombinant protein CPB1 prepared in this study,which showed good precision and high sensitivity,and may be used for the detection of a large number of clinical samples.

关 键 词：产气荚膜梭菌毒素β1 基因重组 原核表达 间接ELISA法 单克隆抗体 

分 类 号：S852.61[农业科学—基础兽医学]