基于牛白血病病毒gp51蛋白建立间接ELISA检测方法  

Establishment of Indirect ELISA Based on Bovine Leukemia Virus gp51 Protein

作　　者：曹丙蕾 刘敏 张群 王飞 王群 CAO Bing-Lei;LIU Min;ZHANG Qun;WANG Fei;WANG Qun(Technology Center of Jinan Customs District,Jinan 250014;Technology Center of Qingdao Customs District,Qingdao 266114)

机构地区：[1]济南海关技术中心,济南250014 [2]青岛海关技术中心,青岛266114

出　　处：《中国口岸科学技术》2021年第11期4-10,共7页China Port Science and Technology

基　　金：国家重点研发计划(2016YFD050110401);济南海关科研项目(2019JK004)。

摘　　要：本研究通过扩增gp51蛋白基因,与pET-32a连接构建重组表达载体,经表达、纯化获得重组gp51蛋白。利用该蛋白为包被抗原,通过优化反应条件,建立牛血清中BLV抗体的间接ELISA方法。SDS-PAGE鉴定表明,成功表达纯化了预期大小为47 kDa的重组gp51蛋白。Western blot鉴定表示该蛋白具有较好的抗原性,与无关血清样本无反应原性。建立的间接ELISA方法中抗原的最佳包被量为200 ng/孔,待检牛血清的最佳稀释比例为1:100,结果表明,该方法具有较好的敏感性和特异性,重复性高,与进口ELISA试剂盒的结果符合率达97.8%以上,具有较好的应用前景。In this study, the recombinant expression vector was constructed by amplifying the gp51 protein gene and connecting it with pET-32 a. The recombinant gp51 protein was obtained by expression and purification. Using the protein as the coating antigen,an indirect ELISA method for BLV antibody in bovine serum was established by optimizing the reaction conditions. SDS-PAGE identification showed that the recombinant gp51 protein with the expected size of 47 kDa was successfully expressed and purified.Western blot showed that the protein had good antigenicity and no reactivity with unrelated serum samples. In the established indirect ELISA method, the optimal coating amount of antigen was 200 ng/well, and the optimal dilution ratio of bovine serum to be tested was 1:100. The results showed that the method had good sensitivity, specificity and high repeatability. The coincidence rate with the results of imported ELISA kit was more than 97.8%, which had a good application prospect.

关 键 词：牛白血病病毒 牛地方流行性白血病 gp51蛋白 表达 ELISA 

分 类 号：S852.65[农业科学—基础兽医学]