小反刍兽疫病毒抗体间接ELISA检测方法的建立

Establishment of indirect ELISA for detection of antibody against peste des petits ruminants virus

为建立一种能快速、特异、敏感地检测血清中小反刍兽疫病毒(PPRV)抗体的方法,通过RT-PCR方法扩增出1 830bp的小反刍兽疫病毒的H基因,并将其克隆至原核表达载体pET-22b(+)中,进行H基因的重组表达。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,建立间接ELISA检测方法。通过矩阵法优化抗原最佳包被浓度、血清稀释度、最佳封闭剂、酶标二抗的最佳稀释度及孵育时间。结果显示,重组H蛋白(分子质量为68ku)能与PPRV阳性血清发生特异性反应。用建立的间接ELISA与标准竞争ELISA试剂盒检测92份临床样品并进行比较,两者的符合率为94.11%。结果表明,本研究建立的间接ELISA可以用于临床PPRV抗体的检测。

To develop a rapid,specific and sensitive method for detection of serum antibodies against peste des petits ruminants virus(PPRV),the PPRV H gene was amplified by RT-PCR.The amplified gene was cloned into pET-22b(+)expression vector and the expressed recombinant protein was detected by SDS-PAGE and Western-blot.Furthermore,indirect ELISA was developed using recombinant PPRV H protein as a coating antigen.Through the matrix method the reaction conditions of the indirect ELISA were optimized,such as coating antigen concentration,dilution of sera,best blocking solution and reaction time of sera.The recombinant H protein with a molecular mass of 68ku was detected by SDS-PAGE,which was recognized by PPRV positive serum in Western-blot.The established indirect ELISA was then used to detect serum antibodies against PPRV.Compared with standard competitive ELISA kit,the coincidence rate between the two methods was 94.11%.The results demonstrated that the developed indirect ELISA performs well in the PPR diagnosis.