摘要
为检测产气荚膜梭菌(Cp)β_(2)毒素抗体,合成产气荚膜梭菌β_(2)毒素基因并插入pET-32a载体,诱导表达后得到Cpβ_(2)毒素重组蛋白,以纯化的Cpβ_(2)毒素重组蛋白为包被抗原,Cpβ_(2)毒素单抗2A4为竞争性抗体与绵羊血清竞争性反应,HRP-山羊抗鼠Ig G为二抗,建立并优化Cpβ_(2)毒素抗体的竞争ELISA检测方法。结果显示,蛋白包被浓度为1.5μg/m L,待检血清不稀释,二抗1∶20000稀释,避光显色15 min为最优反应条件。所建方法的阴性临界值为31.44%,阳性临界值为37.65%,检测阳性血清敏感度为1∶64,批间、批内试验变异系数均小于10%。与Cp的α、β_(1)、ε毒素,腐败梭菌以及大肠杆菌阳性血清均无非特异性反应。检测200份临床绵羊血清样品,该竞争ELISA方法与间接ELISA方法的阳性符合率为85.7%,阴性符合率为95.9%,总符合率为92%。检测100份牛血清,符合率为100%。结果表明,本试验所建方法具有良好的重复性、特异性和敏感性,可以用于检测动物体内是否存在针对Cpβ_(2)毒素的抗体,为评估动物是否感染Cp提供了有效手段,并在Cp的流行病学调查及疫苗免疫效果评价中具有广阔的应用前景。
In order to detect the antibody to Clostridium perfringens(Cp)β_(2)toxin,the Cpβ_(2)toxin gene was synthesized and cloned into pET-32a vector,and the recombinant Cpβ_(2)toxin protein was obtained after induced expression.The purified Cpβ_(2)toxin recombinant protein was used as the coating antigen,Cpβ_(2)toxin monoclonal antibody 2A4 is the primary competitive antibody with sheep serum in competitive reaction,HRP-Sheep anti-mouse IgG is the secondary antibody.A competitive ELISA procedure was developed and refined for the identification of antibodies targeting the Cpβ_(2)toxin.The results of the study revealed that the best reaction conditions were achieved at a protein coating dosage of 1.5μg/mL,the serum to be tested was not diluted,1∶20000 dilution of secondary antibody and 15 min of color development away from light.The negative critical value(PI)is 31.44%,and the positive critical value(PI)is 37.65%,the sensitivity of positive serum detection was 1∶64,and the coefficient of variation of inter-batch and intra-batch tests was less than 10%.The positive sera againstα,β_(1),epsilon of Cp,Clostridium septicum and Escherichia coli were all nonspecific.The positive coincidence rate between the cELISA and iELISA was 85.7%,the negative coincidence rate was 95.9%and the total coincidence rate was 92%.The coin-cidence rate of 100 samples of bovine serum was 100%.The results showed that the proposed method has good repeatability,specificity and sensitivity.It can be used to detect the presence of antibodies against Cpβ_(2)toxin in animals,providing an effective means to assess whether animals are infected with Cp,and has a wide application prospect in the epidemiological investigation of Cp and the evaluation of vaccine immune effect.
作者
赵佳慧
马祎芳
李学瑞
高鹏程
狄娜
郑福英
储岳峰
ZHAO Jiahui;MA Yifang;LI Xuerui;GAO Pengcheng;DI Na;ZHENG Fuying;CHU Yuefeng(State Key Laboratory for Animal Disease Control and Prevention,College of Veterinary Medicine of Lanzhou University,Lanzhou Veterinary Research Institute,Chinese Academy of A gricultural Sciences,Lanzhou 730046,China)
出处
《中国兽医科学》
2025年第8期1026-1032,共7页
Chinese Veterinary Science
基金
国家重点研发子课题:羊梭菌三联四防基因工程亚单位疫苗研发(2023YFD1802504-04)
兰州市科技计划项目(2023-1-14)
中国农业科学院创新工程项目(CAAS-ASTIP-2021-LVRI)。
作者简介
赵佳慧(1999-),女,山东潍坊人,硕士生,研究方向为兽医微生物与免疫学,E-mail:zjh0616zy1@163.com;通讯作者:李学瑞(1971-),男,副研究员,博士,研究方向为动物细菌病的防控技术研究,E-mail:lixuerui@caas。cn;通讯作者:储岳峰(1978-),男,研究员,博士,研究方向为动物细菌病(支原体病)的防控技术基础及应用研究,E-mail:chuyuefeng@caas.cn。
