摘要
为建立绵羊产气荚膜梭菌ε毒素抗体间接ELISA检测方法,对D型产气荚膜梭菌(C60-1株)的ε毒素基因进行扩增,构建pET-32a-ε重组质粒,转入BL21(DE3)PlysS诱导蛋白表达,经Ni柱纯化后获得可溶性ε毒素重组蛋白。以纯化的重组蛋白作为包被抗原,对ELISA反应条件进行优化,并对该方法的特异性、敏感性、重复性和临床检测中的应用进行验证。结果显示,重组蛋白包被浓度为0.5μg/m L,封闭液为1%BSA,待检绵羊血清1∶100稀释,家兔抗山羊HRP酶标二抗1∶5000稀释,显色液避光显色20 min为最佳反应条件。测定45份阴性血清,计算其平均值(X)和标准差(SD),确定所建立的ε毒素抗体间接ELISA方法的阴阳性临界值(X+3SD)为0.254。检测阳性血清敏感度为1∶3200,批间和批内重复率显示,变异系数均小于10%,特异性试验显示,检测羊痘病毒(SPV)、蓝舌病病毒(BTV)、羊布鲁氏菌、沙门菌和绵羊肺炎支原体均没有特异性反应。检测90份临床羊血清样品,该间接ELISA方法与商品化试剂盒之间的阳性符合率为82.9%,阴性符合率为95%,总符合率为85.6%。结果表明,本试验建立的方法具有良好的特异性、敏感性和重复性,可初步用于绵羊产气荚膜梭菌ε毒素抗体的检测。
An indirect ELISA method has been established for testing serum antibody against Clostridium perfringens(C.perfringens)εtoxin in sheep,by amplifying the whole coding sequence ofεtoxin from genome of C.perfringens DC60-1 strain and correctly inserting into expression plasmid pET-32a.The recombinant plasmid was named as pET-32a-ε,which was then transferred into BL21(DE3)PlysS for expressingεtoxin.The recombinantεtoxin protein with soluble form was purified via Ni purification column.Using this recombinant protein as the coating antigen,the reaction parameters of indirect ELISA testing were optimized,and the specificity,sensitivity,reproducibility and application of the method in clinical testing were also validated.In result,the optimal coating concentration of antigen(0.5μg/mL),the blocking solution(1%BSA),the blocking time(1 hour at 37℃),the testing sheep serum dilution(1∶100),the rabbit anti-goat HRP enzyme-labeled secondary antibody dilution(1∶5000).By using 45 samples as negative sera,the average value(X)and standard deviation(SD)were calculated,furthermore the critical value(X+3SD)of the indirect ELISA was established as 0.254.The testing sensitivity for the positive samples was identified as 1∶3200.The repetition rates for inter-assay and intra-assay represented the coefficient of variation with less than 10%.The testing specificity showed no cross-reaction to sheep pox virus,bluetongue virus,Brucella,Salmonella,and Mycoplasma hyopneumoniae in sheep sera.In comparison of this indirect ELISA testing to an commercial kit with 90 clinical serum samples of sheep,the positive coincidence rate was 82.9%,the negative coincidence rate was 95%,and the total coincidence rate was 85.6%.The results indicate that this indirect ELISA method for testingεtoxin antibodies has good specificity,sensitivity and repeatability,which can be preliminarily used for the detection ofεtoxin antibodies of C.perfringens.
作者
王武斌
崔洁文
曹小安
高鹏程
马清龙
李学瑞
储岳峰
WANG Wu-bin;CUI Jie-wen;CAO Xiao-an;GAO Peng-cheng;MA Qing-long;LI Xue-rui;CHU Yue-feng(State Key Laboratory for Animal Disease Control and Prevention,College of Veterinary Medicine of Lanzhou University,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2023年第4期426-433,共8页
Chinese Veterinary Science
基金
西藏自治区中央引导地方项目(XZ202001YD0025C)
甘肃省重点人才项目(2021RCXM047)
中国农业科学院兰州兽医研究创新应用研究项目(110231150007008)
科技重大专项-国家重点实验室重组项目(22ZD6NA001)。
