基于非洲猪瘟病毒p10蛋白间接ELISA检测方法的建立

Establishment of an indirect ELISA method based on African swine fever virus p10 protein

为建立1种快速、高效和高通量检测非洲猪瘟病毒(ASFV)的血清学方法,构建重组质粒pET-28a(+)-p10,通过原核表达系统获得重组p10蛋白,并将其作为包被抗原,构建检测ASFV抗体的间接ELISA方法。结果显示:重组p10蛋白以可溶性和包涵体2种方式表达,分子质量为12 ku,反应原性良好;建立的间接ELISA抗体检测方法最佳反应条件为:1μg/mL抗原4℃包被12 h,50 mg/mL BSA37℃封闭2 h,待检血清孵育0.5 h,底物显色4 min;试验依据S/P值判定阴阳性的临界值为0.388;建立的间接ELISA方法阳性血清的最低检测限为1∶800;与其他常见的猪病阳性血清无交叉反应;批内和批间变异系数均小于10%;与商品化试剂盒的总体符合率为90.38%。研究表明,建立的基于p10表达蛋白的ASFV抗体检测方法具有良好的灵敏性、特异性、重复性和符合率,具有较强的临床应用价值,为ASFV血清学诊断试剂的研发提供了参考。

In order to establish a rapid,efficient and high-throughput serological detection method for African swine fever virus(ASFV),the recombinant plasmid pET-28a(+)-p10 was constructed,and the recombinant p10 protein was obtained through the prokaryotic expression.An indirect ELISA method for the detection of ASFV antibody was established which took the recombinant p10 protein as the coating antigen.The results showed that the recombinant protein was expressed both in soluble and inclusion bodies with a molecular mass about 12 ku,and had good immunoreactivity.The optimal reaction conditions of the indirect ELISA method was as follows:1μg/mL antigen coated at 4℃for 12 h,50 mg/mL BSA blocked at 37℃for 2 h,serum reaction time 0.5 h and the substrate reaction time 4 min.The S/P value was introduced for positive and negative determination,and the critical value was 0.388.The limit of the detection reached 800 times dilution using standard positive serum,the indirect ELISA methodhad no cross-reaction with positive sera of other common swine diseases.The coefficient of variationof the intra-and inter-assay were less then 10%.Compared with commercialized ASFV testing kit,thetotal compliance rate was 90.38%.The above results showed that this method had good sensitivity,specificity,repeatability and consistency rate,and had high clinical application value,which provideda reference for the development of ASFV serological diagnostic reagents.