中国兽医科学2024，Vol.54Issue(10)：1309-1317,9.DOI:10.16656/j.issn.1673-4696.2024.0164
腐败梭菌α毒素蛋白的原核表达及间接ELISA抗体检测方法的建立
Prokaryotic expression of Clostridium septicum alpha toxin protein and establishment of an indirect ELISA method for detection of anti-α toxin antibody
摘要
Abstract
To establish an indirect ELISA method for the detection of the antibodies to Clostridium septicum(CS)α-toxin,the codon-optimized α toxin gene of C.septicum was synthesized and ligated into the pGEX-4T-1 vector and the recombinant plasmid pGEX-4T-1-α was constructed,then the recombinant plasmid was transformed into Escherichia coli BL21(DE3)pLysS for IPTG induced protein expression.Af-ter the recombinant protein was purified and the GST tag was removed,the recombinant CS α-toxin pro-tein was used as the coating antigen to establish an indirect ELISA for detection of anti-CS α-toxin antibody.It was found that the optimized ELISA condition was to coat the recombinant protein at the concentration of 200 ng/well,the dilution ratio of sheep serum was 1∶200,the blocking solution was 10 g/L BSA,the dilution ratio of rabbit anti-goat HRP antibody was 1∶8 000,and the color solution was added and reacted in the dark for 20 min.Totally 50 CS negative serum samples were tested and the mean((x))and standard deviation(s)were calculated.Based on((x)+2s)value,the cut-off value of the established indirect ELISA was 0.244.The sensitivity of serum was 1∶6 400.Both the intra-assay and inter-assay reproducibility results showed that the coefficient of variation was less than 10％.The specificity testing results showed that there was no cross-react with positive sera for Clostridium perfringens type B,Clostridium perfringens type C,Brucella ovis,Salmonella typhimurium and sheeppox virus.A to-tal of 100 clinical serum samples were tested with the established indirect ELISA and a commercial ELISA kits,the results showed that the rate of positive consistent,negative consistent and total con-sistent of our method to the commercial kit was 94.87％,81.97％,87.00％,respectively.The detection results of sheep sera immunized with commercial Clostridium vaccines showed our method is better re-flected the fluctuation of anti-CS α-toxin antibody in immunized sheep sera.This method has good sen-sitivity,repeatability and specificity,and is suitable for the detection of clinical serum samples of C.septicum infection and for the evaluation of the immune effect of corresponding vaccines.关键词
腐败梭菌/α毒素/原核表达/间接ELISAKey words
Clostridium septicum/alpha toxin/prokaryotic expression/indirect ELISA分类
农业科技引用本文复制引用
马清龙,赵佳慧,王武斌,李学瑞,孙雨,李斌,储岳峰..腐败梭菌α毒素蛋白的原核表达及间接ELISA抗体检测方法的建立[J].中国兽医科学,2024,54(10):1309-1317,9.基金项目
兰州市科技计划项目(2023-1-14) （2023-1-14）
国家重点研发计划项目子任务(2023YFD180250404) （2023YFD180250404）
中国农业科学院兰州兽医研究所创新应用研究项目(110231150007008) （110231150007008）
