猪流行性腹泻病毒S蛋白主要抗原表位区的原核表达及间接ELISA检测方法的建立

Prokaryotic expression of the structural protein S of porcine epidemic diarrhea virus and establishment of an indirect ELISA for an detection of antibody against porcine epidemic diarrhea

摘要:

【目的】建立一种快速、特异、敏感的检测血清中猪流行性腹泻病毒(PEDV)抗体的方法。【方法】利用生物学软件对PEDV S蛋白进行抗原位点分析，选择S蛋白的主要抗原表位区进行原核表达。采用SDS-PAGE和Western-blot对重组蛋白进行鉴定及抗原性分析。用纯化的重组蛋白作为包被抗原，经过条件优化、特异性和重复性试验，建立一种针对血清中PEDV抗体的间接ELISA检测方法。【结果】表达了重组S蛋白，重组的S蛋白能与PEDV阳性血清发生特异性反应，并建立一种基于重组S蛋白的间接ELISA检测方法。组内及组间变异系数均小于10%，重复性较好。建立的间接ELISA检测方法分别与商品化PEDV抗体检测试剂盒和western-blot鉴定结果相比，两者符合率分别为86.67%和88.89%。【结论】建立的间接ELISA方法可以用于PEDV抗体的检测。

Abstract:

[Objective] To develop a rapid, specific and sensitive method for detecting the serum antibodies against porcine epidemic diarrhea virus (PEDV). [Methods] The major S protein antigenic region were expressed after analyzing the site antigenic of PEDV S protein by bioinformatics softwares and the recombinant protein was detected by SDS-PAGE and Western-blot. Furthermore, indirect ELISA was developed by using the recombinant protein as coating antigen and though optimizing conditions, specificity and reproducibility tests. [Results] The recombinant protein was expressed successfully, the recombinant S protein could react with PEDV positive serum specifically, and the indirect ELISA method for serum antibodies against PEDV was developed successfully. Both the intro-batch and inter-batch variation coefficient were lower than 10%. The developed indirect ELISA method compared with commercialization of PEDV antibody detection kit and Western-blot, the coincidence rate was 86.67% and 88.89% respectively. [Conclusion] The established indirect ELISA could be used for detecting PEDV antibodies.