梅迪-维斯纳病毒Gag蛋白原核表达与间接ELISA检测方法的建立

Prokaryotic Expression of Maedi-Visna Virus Gag Protein and Development of an Indirect ELISA for Detection of Antibody

Gag蛋白是梅迪-维斯纳病毒(MVV)的主要结构蛋白,也是诊断梅迪-维斯纳病的主要靶蛋白。为建立MVV抗体间接ELISA检测方法,利用大肠埃希菌将含Gag基因的质粒转化到BL21(DE3)感受态细胞中,最终获得高效表达的Gag蛋白,进一步进行ELISA条件的优化。所建立的方法敏感性试验显示在阳性血清1∶3 200倍稀释时仍能检出,其组内与组间变异系数均低于10%,具有良好的重复性。对162份临床血清样品进行检测,同时用购自法国IDVET公司的间接ELISA进行比较,符合率达到89.65%。应用该方法对新疆地区1个种羊场和1个育肥羊场的294份羊血清样品进行了检测,结果显示MVV抗体阳性率分别为10.5%和1.77%,表明种羊场中MVV感染率高于育肥羊场,该研究结果可为MVV抗体检测试剂盒的开发提供支持。

Maedi-Visna virus(MVV)Gag protein is the major structural protein of virus particles,and it is the main target protein in the diagnosis of MVV.In order to establish an indirect ELISA detection method for MVV antibody,Escherichia coli was used to transform the plasmid containing Gag gene into BL21(DE3)competent cells,and finally Gag protein with high expression was obtained,and the ELISA conditions were further optimized.The results of sensitivity test indicated that the detection limit for positive serum was 1:3200.The results of reproducibility test showed that the coefficient of intra-variations and inter-variations were less than 10%.Compared with the IDVET kit for detection of MVV antibodies,the total positive rate of the indirect ELISA developed in this study was very high,with a coincidence rate of 89.65%.Using this method to the breeding sheep and fattening sheep in Xinjiang region,294seral samples were tested.The results showed that the positive rates of antibody against MVV were 10.50%,1.77%respectively.The method was of great practical significance for detection of MVV antibodies in sheep farms.