非洲猪瘟病毒P30蛋白的真核表达及间接ELISA抗体检测方法的建立

Eukaryotic expression of P30 gene of African swine fever virus and establishment of indirect ELISA for antibody detection

为建立快速检测猪血清中非洲猪瘟病毒(ASFV)抗体的间接ELISA方法,本研究以ASFV Pig/HLJ/2018株基因组为模板优化合成CP204L基因(1 bp~549 bp),构建pFastbac1-P30-His重组表达载体,利用杆状病毒-昆虫细胞真核表达系统表达ASFV重组P30蛋白(rP30),以Ni亲和纯化后的rP30作为包被抗原,经过一系列条件优化,建立了一种快速检测ASFV P30抗体的血清学方法。结果显示,胞外分泌的rP30约为30 ku,western blot鉴定结果显示其具有良好的反应原性;经优化确定了ELISA最佳反应条件:包被量为0.4μg/mL,待检血清稀释倍数为1.25,血清反应时间为30 min,兔抗猪IgG-HRP稀释度为1.10000。利用该方法同时检测猪临床常见病毒阳性血清,结果显示建立的ASFV抗体间接ELISA方法仅对ASFV感染血清检测为阳性,与猪瘟病毒、猪伪狂犬病病毒、猪繁殖与呼吸综合症病毒、猪圆环病毒和猪口蹄疫病毒阳性血清均无交叉反应,具有较强的特异性;将ASFV感染血清2倍倍比稀释后,利用本实验建立的ASFV抗体间接ELISA方法、ID.VET(P32)试剂盒和INGENASA(P72)试剂盒同时检测,结果显示本实验建立的方法检测灵敏度为1.128,与INGENANA(P72)试剂盒相同,低于ID.VET(P32)试剂盒(1.1024);批内和批间重复性试验的变异系数均小于10%,重复性良好。利用该方法与ID.VET试剂盒同时对383份临床样品进行检测,二者符合率为98.9%。本研究建立的方法可为ASF的监测和防控提供技术支持。

In order to establish an indirect ELISA method for detecting African swine fever virus(ASFV)antibodies in swine serum,CP204 L gene(1-183 aa)from the ASFV Pig/HLJ/2018 strain was optimized synthesis and cloned into pFastbac1-Signal-His vector.Subsequently the bac-to-bac expression system was used to express the ASFV P30 protein,and the ASFV P30 recombinant protein was purified by Ni affinity and used as the coating antigen.After a serological conditions optimization,a rapid detection of the antibody against ASFV P30 was established.The results showed that the recombinant protein was approximately 30 ku expressed in culture supernatant,and showed good reactogenicity by western blot identification.The optimum reaction conditions of P30-ELISA were determined as follows:antigen coating concentration was 0.4μg/mL,serum dilution was 1.25,serum incubation time was 30 min,and secondary antibody dilution was 1.10000.The P30-ELISA method had good specificity against ASFV and no cross-reaction with positive sera of classical swine fever virus,pseudorabies virus,porcine reproductive and respiratory syndrome virus,porcine circovirus and foot-and-mouth disease virus.The ASFV positive serum was performed by 2-fold serial dilution and simultaneous detected by the ASFV antibody indirect ELISA method established in this study,ID.VET(P32)kit and INGENANA(P72)kit.The limit of the detection reached 1.128 using standard positive serum,which is the same as the INGENANA kit but lower than the ID.VET kit(1.1024).The inter-and intra-assay coefficient of variation(CV)were both lower than 10%,which showed good repeatability.The coincidence rate of the detection results of clinical 383 samples between the P30-ELISA method and ID.VET kit was 98.9%.The method established in this study can provide technical support for ASF surveillance and control.