中国兽医科学2017，Vol.47Issue(5)：563-568,6.DOI:10.16656/j.issn.1673-4696.2017.05.005
山羊关节炎-脑炎病毒抗体间接ELISA检测方法的建立
Development of an indirect ELISA for the detection of antibodies against caprine arthritis encephalitis virus using recombinant CA protein
摘要
Abstract
In order to develop an indirect ELISA for the detection of antibodies against caprine arthritis encephalitis virus(CAEV) using recombinant CA protein,the CA gene was amplified by polymerase chain reaction,and then the amplified DNAfragment was cloned into an expression vector,pET-28a(+).The recombinant bacterial(BL21) cells harboring plasmid pET-28a(+)-CA were induced to express recombinant protein rHis-AC.The recombinant protein rHis-AC was purified by affinity chromatography using Ni-NTA column.The rHis-CA ELISA for detection of antibodies against CAEV was determined by optimazing conditions using purified rHis-AC as coating antigen.The results showed that the rHis-CA was expressed as inclusion bodies and had good reactionogenicity.The optimal concentration of coating antigen and dilution of serum were 0.4 μg/mL and 1∶100,respectively.The cut-off value of negative and positive sera was 0.45.The rHis-AC ELISA method exhibited good specificity,and showed no cross-reactions with positive sera of sheeppox virus,bluetongue virus and foot-and-mouth disease virus.0f the 48 goat serum samples from Shaanxi,35.41％(17/48) were positive in rHis-CA ELISA and 31.25％were positive in agar gel immunodiffusion(AGID).All 240 goat serum samplesfrom Tianjin were negative by rHis-CAELISAandAGID.关键词
山羊关节炎-脑炎/重组CA蛋白/间接酶联免疫吸附试验Key words
caprine arthritis encephalitis/recombinant CA protein/indirect ELISA分类
农业科技引用本文复制引用
王建华,黄金海,孙越辉,陈小金,王玉玲,肖妍,王乃福,陈本龙,董志珍,赵祥平..山羊关节炎-脑炎病毒抗体间接ELISA检测方法的建立[J].中国兽医科学,2017,47(5):563-568,6.基金项目
国家质检总局科技计划项目(2011IK016) （2011IK016）
