罗非鱼源无乳链球菌ScpB蛋白的表达及IgM抗体间接ELISA方法的建立

Cloning and expression of scpb protein of Streptococcus agalactiae from Tilapia and establishment of indirect ELISA for IgM antibody detection

为了建立一种具有良好特异性、敏感性、重复性和准确性的,能够检测罗非鱼源无乳链球菌的ELISA方法,试验首先进行了罗非鱼源无乳链球菌ScpB基因的克隆及其蛋白的表达和纯化,然后以重组蛋白作为包被抗原、罗非鱼待测血清作为一抗、抗罗非鱼IgM单克隆抗体2C10作为二抗、羊抗鼠IgG-HRP作为三抗建立间接ELISA方法并对该方法进行条件(抗原包被质量浓度、一抗稀释度、抗原包被条件、封闭液、一抗作用时间、二抗作用时间)优化,最后确定该方法的临界值并进行特异性、敏感性、重复性和符合性试验。结果表明:纯化后的罗非鱼源无乳链球菌ScpB蛋白大小为104.6 ku,与预期大小一致,可被罗非鱼IgM抗体特异性识别;建立的IgM抗体间接ELISA方法的最佳抗原包被质量浓度为0.1μg/mL,最佳一抗稀释度为1∶5,最佳抗原包被条件为4℃、12 h,最佳封闭液为5%脱脂乳,最佳一抗作用时间为2 h,最佳二抗作用时间为0.5 h;临界值上限为0.456,临界值下限为0.377;批内变异系数为3.349%~5.272%,批间变异系数为2.793%~5.902%,均低于10.000%;仅可检测出罗非鱼无乳链球菌阳性血清和罗非鱼ScpB蛋白免疫血清,无法检测出罗非鱼嗜水气单胞菌阳性血清、罗非鱼荧光假单胞菌阳性血清、罗非鱼海豚链球菌阳性血清和罗非鱼鳗弧菌阳性血清;待测血清最大稀释度为1∶640;与无乳链球菌全菌包被ELISA检测方法比较,阳性率略低,符合率为83.43%。说明本研究建立的罗非鱼源无乳链球菌IgM抗体间接ELISA方法具有良好的特异性、敏感性、重复性和准确性。

In order to establish an ELISA method with good specificity,sensitivity,repeatability and accuracy for detecting Streptococcus agalactis from tilapia,firstly,the ScpB gene of Streptococcus agalactiae from Tilapia was cloned,expressed and purified.Then,the indirect ELISA method was established by using the recombinant protein as the coating antigen,with the Tilapia serum for test as the primary antibody,the anti-tilapia IgM monoclonal antibody 2C10 as the secondary antibody,and the goat anti-mouse IgG-HRP as the tertiary antibody.The conditions of the method(antigen coating mass concentration,primary antibody dilution,antigen coating conditions,blocking solution,primary antibody action time,secondary antibody action time)were optimized.Finally,the critical value of the method was determined and the specificity,sensitivity,repeatability and compliance tests were carried out.The results showed that the size of the purified ScpB protein of Streptococcus agalactiae from Tilapia was 104.6 ku,which was consistent with the expected size and could be recognized by Tilapia IgM specific antibody.The optimal antigen-coated mass concentration of the established IgM antibody indirect ELISA method was O.1μg/mL;the optimal primary antibody dilution was 1:5;the optimal antigen-coated condition was 4℃for 12 h;the optimal sealing solution was 5%skim milk powder;the optimal primary antibody action time was 2 h and the optimal secondary antibody action time was 0.5 h.The upper limit of critical value was 0.456,and the lower limit of critical value was 0.377.The intra-assay coefficient of variation was 3.349%-5.272%,and the inter-assay coefficient of variation was 2.793%-5.902%,both of which were lower than 10.000%.The positive serum of Streptococcus agalactiae from Tilapia and the immune serum of Tilapia ScpB protein could only be detected,but the positive serum of Aeromonas hydrophila from Tilapia,the positive serum of Pseudomonas fluorescens from Tilapia,the positive serum of Streptococcus iniae from Tilapia and the positive serum of Vibrio angullarum from Tilapia could not be detected.The maximum dilution of the tested serum was 1:640.The positive rate was slightly lower than that of the whole Streptococcus agalactiae coated by ELISA,and the coincidence rate was 83.43%.The results indicated that the Streptococcus agalactia IgM antibody from Tilapia indirect ELISA method had good specificity,sensitivity,repeatability and accuracy.