可溶性E2蛋白作为抗原的检测猪瘟病毒血清抗体间接ELISA方法的建立

以可溶性重组E2蛋白作为抗原,建立了猪瘟病毒(CSFV)血清抗体间接ELISA诊断方法(rE2-ELISA).将猪瘟疫苗毒株E2基因主要抗原区(A-D)基因克隆到表达栽体pGEX-6p-1上,转化E.coli,降低诱导温度至20℃,获得48 000大小E2融合蛋白,部分目的蛋白以可溶性形式表达.Western blotting试验证实,E2融合蛋白可以和CSFV阳性血清发生特异性结合.亲和层析纯化后的E2融合蛋白作为抗原,建立了检测CSFV血清抗体的间接rE2-ELISA方法.该方法的特异性试验结果表明,与PRRSV、PCV2、PPV和PRV阳性血清之间不存在交叉反应;用rE2-ELISA和国外同类试剂盒(CSF-Ab-Kit)检测142份田问血清样品,2种试剂盒的阳性检测率分别为83.81%和88.73%.因此,rE2-ELISA猪瘟抗体检测试剂盒具有良好的敏感性和特异性,适合应用在大规模的CSFV血清抗体的检测工作中.

An indirect ELISA using a soluble protein E2 as antigen for detection of antibodies to classical swine fever virus

An indirect ELISA,based on soluble E2 recombinant protein of classical swine fever virus,was established for the detection of antibodies against CSFV.The antigenic regions(A-D)of structural protein E2 of the C-strain was cloned and constructed into expressive vector pGEX-6p-1.A portion of soluble E2 fusion protein,approximate to 48ku,was expressed at 20℃ in E.coli.The antigenicity of this recombinant protein was demonstrated by western blotting and purified by using glutathione sepharose 4B resin affinity chromatography.The purification of E2 protein was as an antigen in an indirect ELISA for the detection of the antibodies to CSFV.Cross-reactivity assay showed that this assay was CSFV-specific,and compared with the CSFV-Ab-kit,the detectivity of the positive sera in 142 field serum samples was 83.81% and 88.73%,respectively,indicating that no significant difference between the two assays.The rE2-ELISA has highly specific and sensitivity when applied to test sera.Therefore,it is suitable for large scale monitoring of CSF.