绵羊梅迪-维斯纳病毒CA蛋白可溶性表达、多克隆抗体制备及鉴定

Soluble Expression of CA Protein of Maedi-visna Virus of Sheep Origin and Preparation and Identification of Its Polyclonal Antibody

[目的]制备绵羊梅迪-维斯纳病毒(maedi-visna virus,MVV)衣壳蛋白(capsid protein,CA)多克隆抗体,并鉴定其特异性。[方法]根据MVV内蒙古分离株CA基因序列设计特异性引物,扩增CA基因,构建重组质粒;对CA重组蛋白进行原核表达及纯化,制备兔源MVV CA重组蛋白多克隆抗体,采用间接ELISA方法测定其抗体效价,利用Western blot和免疫组化方法对其进行特异性鉴定。[结果]成功构建MVV CA重组蛋白原核表达系统,纯化后的目的蛋白大小约27 kDa;间接ELISA方法测定制备的多克隆抗体效价为1∶8192;Western blot检测感染MVV绵羊的病肺组织,在25 kDa处出现特异性条带;免疫组化结果显示感染MVV绵羊的病肺中巨噬细胞的胞浆内有明显棕黄色阳性信号。[结论]利用获得的可溶性重组MVV CA蛋白制备的多克隆抗体具有较好特异性,可为MVV血清学诊断技术提供检测抗体。

[Objective]The aims of this study were to prepare the polyclonal antibody against capsid(CA)protein of a maedi-visna virus(MVV)strain isolated from naturally infected sheep and to assess its specificity.[Method]A set of specific primers was designed according to the CA gene sequence of MVV Inner Mongolia strain,and the CA gene was subsequently amplified by PCR assay for constructing a recombinant plasmid.The MVV CA recombinant protein was prokaryotically expressed and purified.The rabbit-derived polyclonal antibody against MVV CA recombinant protein was prepared,and its titer and specificity were determined and identified by using indirect ELISA assay as well as Western blot and immunohistochemical analyses.[Result]The prokaryotic expression system of MVV CA recombinant protein was successfully constructed,and the target protein was about 27 kDa after purification.The titer of the prepared polyclonal antibody was determined as 1∶8192 by indirect ELISA assay.Western blot analysis showed that there was a specific band with a size of 25 kDa in lung tissues of sheep infected with MVV.Immunohistochemical analysis demonstrated that there were obvious brownish yellow positive signals in cytoplasm of macrophages in lung tissues of sheep infected with MVV.[Conclusion]Polyclonal antibody prepared with soluble recombinant MVV CA protein had good specificity,which might serve as a candidate antibody in MVV serological diagnosis technology.