非洲猪瘟病毒p72蛋白间接ELISA抗体检测方法的建立

Prokaryotic expression of African swine fever p72 protein and development of indirect ELISA method

为建立非洲猪瘟病毒(ASFV)的血清学检测方法,本研究根据ASFV Georgia2007/1株基因组合成其p72全长基因,并将其克隆至p GEX6p-1载体,构建重组质粒pGEX6p-1-p72并经酶切、PCR和测序鉴定正确后转化BL21感受态细胞,经IPTG诱导表达后经SDS-PAGE和western blot鉴定,结果显示获得了可溶性表达且纯度较高的重组p72蛋白(rp72)。利用GST亲和纯化层析柱纯化,得到浓度为0.2 mg/mL的rp72。以rp72作为包被抗原,通过优化各反应条件建立检测猪血清中非洲猪瘟抗体的间接ELISA方法,结果显示,rp72最佳包被浓度为1.25μg/mL,封闭时间为37℃1 h;待检血清稀释度为1.100,孵育时间为37℃30 min;羊抗猪IgG-HRP孵育时间为37℃1 h。利用该方法检测280份阴性血清,确定其临界值为0.412。采用建立的间接ELISA抗体方法对PEDV、PRRSV、CSFV、PRV、TGEV、PPV、PCV、E.coli阳性猪血清进行检测,结果显示,除ASFV阳性对照为阳性外,其他病原阳性血清检测结果均为阴性,该方法特异性强;将非洲猪瘟抗体阳性猪血清2倍倍比稀释(1.100~1.6400)后,利用该方法进行检测,结果显示其检测阳性血清的稀释度为1.3200倍时OD_(450nm)值仍大于临界值,敏感性高于市售西班牙ASFV阻断ELISA试剂盒检测结果,本研究建立的方法敏感性较高;对该方法进行批内和批间重复性试验,结果显示批内和批间的变异系数均<10%,重复性良好。利用本研究建立的ASFV ELISA抗体检测方法(p72)对184份临床血清进行检测,结果显示该方法敏感性为100%,特异性为98.70%;与ASFV阻断ELISA抗体检测试剂盒方法总符合率为98.91%,kappa值为0.961。本研究首次基于rp72建立的非洲猪瘟抗体间接ELISA检测方法具有良好的重复性、敏感性和特异性,为ASFV抗体检测提供了一种快速、精准、经济、高效的方法。

In order to develop a serological assay for the detection of African swine fever virus(ASFV),the full-length ASFV p72 gene was synthesized and cloned into p GEX6 P-1 vector to construct the recombinant plasmid p GEX6 P-1-p72.After identification by restriction enzyme digestion,PCR and sequencing,the recombinant p72 protein was expressed by prokaryotic system(BL21),and identified by SDS-PAGE and western blot assay.The results showed that a soluble and high purity recombinant p72 protein was obtained and the concentration was 0.2 mg/m L,after purification by GST affinity purification chromatography column.The purified p72 protein was used as the coating antigen,and the reaction conditions were optimized to develop an indirect ELISA method for detecting the serum antibodies against ASFV.The optimized reaction conditions were as follows:the optimal coating concentration of antigen was 1.25μg/m L,the blocking condition was 37℃for 1 h;the serum dilution was 1:100,the incubation condition was 37℃for 30 min;and sheep anti-swine Ig G-HRP was incubated at 37℃for 1 h.280 ASFV negative serum samples were detected by this method and the results showed the cut-off value was 0.412.Furthermore,this indirect ELISA method could specifically detect ASFV positive antibodies with no cross reaction with positive sera of other swine viral pathogens,such as PEDV,PRRSV,CSFV,PRV,TGEV,PPV,PCV,BL21(E.coli).In addition,ASFV positive antibody serum was multiple diluted(1:100-1:6400),followed by indirect ELISA detection.The results confirmed that OD_(450nm) value still higher than cut-off value when positive serum sample with 3200-fold dilution,and the sensitivity was higher than commercial Spanish blocking kits.These results suggested that the indirect ELISA method established in this study showed high sensitivity.The coefficients of variation(CV)of the intra-and inter-batch repetitive tests were less than 10%,which showed this method had good repeatability.184 clinical serum samples were tested in this study,and the coincidence rate and kappa value of this method was 98.91%and 0.961,respectively,when compared with the imported commercial kit.All these results suggest that the indirect ELISA method developed basing on the recombinant p72 protein of ASFV in this study possess the features of specificity,sensitivity and repeatability.It provides a rapid,accurate,economical and efficient method for the detection of ASFV antibody in swine serum samples.