产气荚膜梭菌β毒素抗体间接ELISA检测方法的建立  

Establishment of an indirect enzyme-linked sorbent assay for detection of serum antibody of beta-toxin protein of Clostridium perfringens

作　　者：叶宝宏[1] 鲍长磊 付明哲[2] 许信刚[2] YE Baohong BAO Changlei FU Mingzhe XU Xingang(School of Life Science of Yulin Uniwersity ,Yulin ,Shaanxi 719000,China College of Veterinary Medicine,Northwest A ＆ F Uniwersity , Yangling , Shaanxi 712100,China)

机构地区：[1]榆林学院生命科学学院,陕西榆林719000 [2]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2016年第8期55-60,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：陕西省科技攻关项目(2015NY162);西北农林科技大学试验示范站(基地)科技成果推广项目(TGZX2015-32)

摘　　要：【目的】建立产气荚膜梭菌β毒素抗体间接ELISA检测方法。【方法】将原核表达重组质粒pET-28a-β转化大肠杆菌BL21（DE3）感受态细胞,用IPTG进行诱导表达,表达产物经镍柱亲和层析纯化、尿素梯度透析复性后进行Western blot鉴定。用复性的β毒素重组蛋白作为包被抗原,通过优化间接ELISA试验条件,建立其抗体间接ELISA检测方法,对该方法的特异性、敏感性和重复性进行考察,并用其对521份临床样本进行检测。【结果】通过诱导、纯化和复性,获得了纯度较高且具有良好反应原性的β毒素重组蛋白。间接ELISA条件为：抗原最佳包被质量浓度为5.0μg/mL,阳/阴性血清最佳稀释度为1∶50,酶标二抗最佳稀释度为1∶2 500,以含50g/L脱脂奶粉的PBST作为封闭液,37℃封闭1h,抗原抗体作用1h,TMB显色30min。间接ELISA的判定标准为OD450≥0.313为阳性,OD450〈0.313为阴性;建立的间接ELISA方法的特异性为96%,敏感性为98%;批内变异系数在3%-4%,批间变异系数在9%以下。利用间接ELISA检测方法对521份临床山羊血清样本的检测结果表明,抗体阳性率为72.5%。【结论】对产气荚膜梭菌β毒素进行了原核表达,成功建立了其抗体间接ELISA检测方法,为产气荚膜梭菌β毒素抗体检测试剂盒的研制奠定了基础。【Objective】This study was conducted to establish an indirect enzyme-linked immune sorbent assay（ELISA）to detect the serum antibody levels of beta-toxin protein.【Method】The prokaryotic expression plasmids pET-28a-βcontaining beta-toxin gene was successfully transformed into E.coli BL21（DE3）before being induced by IPTG.The expressed protein was purified by Ni-NTA affinity chromatography and analyzed by SDS-PAGE.The purified protein was refolded by urea gradient dialysis,and its specificity was tested by Western-blot.The refolded beta-toxin recombinant protein was taken as coating antigen to determine the best reaction conditions by optimizing the indirect ELISA conditions.An indirect ELISA to detect the serum antibody of beta-toxin protein was then established and its specificity,sensitivi-ty and repeatability were detected.【Result】The optimal conditions of ELISA were：coating concentration of beta-toxin fusion protein was 5.0μg/mL,the serum was diluted by 1∶50,the enzyme labeled antibody was diluted by 1∶2 500,using 50g/L skim milk powder in PBST as sealing fluid for 1hat 37 ℃,and the reaction time of substrate was 30 min.The cut-off value of the assay was 0.313,OD450≥0.313 means positive while OD450〈0.313 means negative.The specificity and sensitivity were 96% and 98%,respectively.The intra-assay and inter-assay variation coefficients were 3%-4%and〈9%,respectively.A Total of 521 sera samples were detected and the positive rate was 72.5%.【Conclusion】The beta-toxin fusion protein was obtained successfully.The establishment of an indirect ELISA provides solid foundation for development of ELISA kit to detect antibodies against Clostridium perfringens beta-toxin antibodies.

关 键 词：产气荚膜梭菌 Β毒素 原核表达 间接ELISA 

分 类 号：S855.1[农业科学—临床兽医学]