重组小反刍兽疫病毒H蛋白的真核表达与间接ELISA检测方法的建立

Expression of recombinant H protein of peste des petits ruminants virus in insect cells and development of an indirect ELISA for detection antibodies against the virus

本研究利用昆虫杆状病毒表达系统成功表达了小反刍兽疫病毒(PPRV)重组H蛋白,并以该H蛋白为包被抗原建立了检测PPRV抗体的间接ELISA方法。通过检测已知背景的绵羊血清,评价了间接ELISA的特异性、敏感性和重复性。结果显示,该ELISA对其他相关的羊类病原无交叉反应,组内与组间变异系数均低于10%。利用建立的I-ELISA法和血清中和试验分别对550份绵羊临床样本进行检测,结果显示,Ⅰ-ELISA法检测的阳性率为76.55%,血清中和试验检测的阳性率为76.73%,敏感性符合率为98.58%;Ⅰ-ELISA法检测的阴性率为23.45%,血清中和试验检测的阴性率为23.27%,特异性符合率为96.09%,两种方法的总符合率为98%。上述结果表明,本研究建立的检测小反刍兽疫病毒血清中和抗体的间接ELISA方法与血清中和试验比较,具有良好的特异性、敏感性和重复性,可开发为临床适应的检测试剂盒。

In this study,recombinant H protein was successfully expressed by insect baculovirus expression system,and an indirect ELISA method for detecting PPRV antibody was established with H protein as coating antigen.The specificity,sensitivity and repeatability of indirect ELISA(Ⅰ-ELISA)were evaluated by detecting sheep serum with known background.The results showed that Ⅰ-ELISA method had no cross reaction to other related sheep pathogens,and the coefficient of variation within and between groups was less than 10%.About 550 clinical samples were tested by the establishedⅠ-ELISA and serum neutralization test respectively.The results showed that the positive rate ofⅠ-ELISA was 76.55%,the positive rate of serum neutralization test was 76.73%,the sensitivity coincidence rate was 98.58%,the negative rate of I-ELISA was 23.45%,the negative rate of serum neutralization test was 23.27%,the specificity coincidence rate was 96.09%,and the total coincidence rate of the two methods was 98%.It is confirmed that the I-ELISA method for detecting serum neutralizing antibody of PPRV established in this study has good specificity,sensitivity and repeatability compared with serum neutralization test,and can be developed as a detection kit for clinical application.