K亚群禽白血病病毒抗体间接ELISA检测方法的建立  

Indirect ELISA for detection of antibodies against subgroup K avian leukosis virus

作　　者：袁丽霞 饶明章 张杰 赵子君 陈建 陈杨一骏 严立福 郑晓翠 谢暮晴 曹伟胜 

机构地区：[1]华南农业大学兽医学院农业部兽用疫苗创制重点实验室,广东广州510642

出　　处：《中国兽医科学》2017年第11期1349-1356,共8页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31672552);广东省科技计划项目(2015A020209145);广东省自然科学基金项目(2016A030313403);广东省家禽产业技术体系疾病控制岗位专家项目(2016LM1114)

摘　　要：为了研究K亚群禽白血病病毒抗体间接ELISA检测方法,以ALV-K GDFX0603株为模板,扩增其gp85基因,构建原核表达质粒p ET-GDFX0603-gp85,筛选原核表达菌液的诱导条件,采用Ni柱纯化表达的重组蛋白;以纯化后重组蛋白作为包被抗原,研究ALV-K抗体间接ELISA检测方法。结果显示,表达的gp85重组蛋白以包涵体形式存在,大小为53 ku。Western-blot分析表明,该蛋白具有良好的免疫反应性;重复性试验结果显示,板内重复性的CV最大值为10.41%;板间重复性的CV最大值为12.57%,CV值均小于15%。特异性试验结果显示,该方法除了对ALV-A阳性血清有弱的交叉反应外,对ALV-B、ALV-J、AIV、NDV、IBDV、MDV、REV、大肠杆菌和沙门菌的阳性血清均无交叉反应。结果表明,初步建立了检测ALV-K抗体的间接ELISA方法,该方法具有良好的板内和板间重复性及较强的特异性。To develop an indirect ELISA method for the detection of antibodies against subgroup K avian leukemia virus,the gp85 gene of ALV-K GDFX0603 strain was amplified by PCR,then the prokaryotic expression plasmid p ET-GDFX0603-gp85 was constructed.The induction conditions of prokaryotic expression were screened,then the recombinant protein was purified by Ni colum.Using purified recombinant protein as coating antigen,an indirect ELISA method for the detection of antibodies against ALV-K was determined.The results showed that the gp85 recombinant protein was expressed as inclusion bodies,and its size was 53 ku.Western-blot analysis showed that recombinant protein had good immunogenicity.The intra-and inter-assay demonstrated that the coefficient of maximum variation was 10.41% and12.57%, respectively.The specific tests showed that there were no cross reactions with the sera against ALV-B,ALV-J,AIV,NDV,IBDV,MDV,REV,Escherichia coli and Salmonella,while it had weak cross reactions with the serum against ALV-A.The results showed that the indirect ELISA method for the detection of antibodies against ALV-K had good reproducibility and better specificity.

关 键 词：K亚群禽白血病病毒 gp85蛋白 原核表达 间接ELISA 

分 类 号：S852.5[农业科学—基础兽医学]