小反刍兽疫病毒重组抗原间接ELISA检测及初步应用

为了建立一种快速、特异、敏感的小反刍兽疫病毒（peste des petits ruminants virus,PPRV）血清学检测方法,本研究将PPRV血凝蛋白（H）和核蛋白（N）克隆至pET28a（＋）载体进行诱导表达,并将2种蛋白Ni柱纯化后包被ELISA反应板,建立起检测PPRV特异性抗体的间接ELISA法。结果表明在E.coli BL21（DE3）中表达出2种PPRV蛋白,将2种重组蛋白纯化后按照1：1比例混合,制备PPRV鸡尾酒抗原,成功建立了特异性强、敏感性高的PPRV间接ELISA法;最后用建立的方法对新疆伊犁、塔城、阿勒泰和喀什边境地区采集的325份山羊、789份绵羊和132份牛血清进行了抗体检测,检测结果显示均为阴性,提示在我国新疆边境地区反刍动物尚未发生PPRV感染,但需要及时进行免疫接种以建立预防PPRV从国外传入的免疫带。本研究建立的PPRV间接ELISA法为该病毒的检测提供基础,也为该病的科学防控提供参考。

Development and Preliminary Application of Indirect ELISA Based on PPRV Recombinant Antigen Protein

In order to develop a rapid,specific and sensitive detection method for peste des petits ruminants virus （PPRV）,in this research,haemagglutinin （H） protein and nucleoprotein （N） protein of PPRV were respectively cloned into pET-28a （＋） for inducement expression,and recombinant proteins were purified by Ni2＋ column to prepare coating antigen to establish an indirect ELISA for PPRV specific antibody.The result showed that recombinant protein H and N of PPRV were respectively expressed in E.coli BL21 （DE3）.Then the cocktail antigen was prepared with this two purified proteins in proportion to 1：1,and then a rapid,specific and sensitive ELISA was successfully developed by using the cocktail antigen to coat ELISA plates.Finally,the serum samples of 325 goats,789 sheep,132 cattle collected from border areas Ili,Tacheng,Altay and Kashi of Xinjiang were assayed with this established indirect ELISA,respectively.The detecting results displayed that all samples sera were negative,which signified that there was no PPRV infection in goats,sheep and cattle in the border area of Xinjiang.However,it is imperative to carry out inoculation to prevent PPRV from foreign countries.The indirect ELISA method of PPRV establishment in this investigetion would provide a basic for detecting this virus as well as a referrence for preventing scientifically.