猪圆环病毒2型ORF2基因截断表达及ELISA抗体检测方法的初步建立  

Dvelopmentof an indirect ELISA based on prokaryotic expression of truncated ORF2 gene of porcine circovirus type 2

作　　者：郭官鹏[1] 刘畅[1] 陈龙彪[1] 马世杰[1] 高晓云[1] 陈红英[1] 崔保安[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002

出　　处：《中国兽医学报》2014年第7期1059-1064,共6页Chinese Journal of Veterinary Science

基　　金：河南省重大科技专项(111100110300)

摘　　要：为建立以重组PCV2Cap蛋白为包被抗原的间接ELISA检测方法,构建了猪圆环病毒2型(PCV2)ORF2基因原核表达质粒pET28a-ORF2。SDS-PAGE显示,在0.1mmol/L IPTG和37℃条件下诱导4h,重组Cap蛋白高效表达。Western blotting证实该蛋白能够被PCV2阳性血清特异性识别。以纯化的蛋白为抗原建立了检测PCV2抗体的间接ELISA方法。结果表明,抗原最适包被质量浓度为2mg/L;血清最佳稀释度为1∶100;酶标二抗最适浓度为1∶2 000,该方法的敏感性为86.96%,特异性为100%。用该方法对河南省152份猪血清样品进行检测,与间接免疫荧光(IFA)的符合率为85.53%(130/152),与商品化的韩国金诺PCV2ELISA试剂盒的符合率为88.16%(134/152)。本试验成功建立了PCV2血清抗体间接ELISA方法,具有较高的敏感性和特异性,可用于大规模的血清学检测。The objective of this research is to establish an indirect ELISA method by using recombinant Cap protein of PCV2as antigen.The recombinant prokaryotic expression plasmid pET28aORF2 was transformed into E.coli BL21（DE3）bacteria and induced by IPTG,SDS-PAGE showed that Cap protein was expressed in the form of inclusion,molecular weight is about 28 000.Based on optimizing expression condition,the largest amount of Cap protein was induced by 0.1mmol/L IPTG at 37℃for 4hours.Western-blot analysis showed that the expression of Cap protein can be specifically recognized by positive serum of PCV2.The purified Cap protein was used as antigen to establish indirect ELISA for detecting PCV2antibodies,the optimal working concentration of Cap protein as coating antigen was 2mg/L,the best dilution of serum and enzyme combined antibodies were 1∶100and 1∶2 000,respectively.The sensitivity and specificity of this method were 86.96%and 100%.Comparing this assay with the indirect immunofluorescence（IFA）and PCV antibody ELISA kit by detecting 152serum samples,results showed that the agreement of them was 85.53%（130/152）and 88.16%（134/152）,respectively.This experiment successfully established indirect ELISA for detecting antibody of PCV2with high sensitivity and specificity that can be used for serological detection of large-scale.

关 键 词：猪圆环病毒2型 CAP蛋白 原核表达 蛋白纯化 间接ELISA 

分 类 号：S852.65[农业科学—基础兽医学]