| 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5.0 μg/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50 g/L脱脂奶粉的PBST作为封闭液，37 ℃封闭1 h，抗原抗体作用1 h，TMB显色30 min。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。 |
| 关键词: 产气荚膜梭菌 β毒素 原核表达 间接ELISA |
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| 基金项目: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） |
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| Establishment of an indirect enzyme-linked sorbent assay for detection of serum antibody of beta-toxin protein of Clostridium perfringens |
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YE Baohong,BAO Changlei,FU Mingzhe,et at
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| Abstract: |
| 【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-β containing 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,sensitivity and repeatability were detected.【Result】The optimal conditions of ELISA were:coating concentration of beta-toxin fusion protein was 5.0 μg/mL,the serum was diluted by 1∶50,the enzyme labeled antibody was diluted by 1∶2 500,using 50 g/L skim milk powder in PBST as sealing fluid for 1 h at 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. |
| Key words: Clostridium perfringens beta-toxin prokaryotic expression indirect ELISA |
