非洲猪瘟病毒p35蛋白作为诊断抗原的抗原性比较

Comparison of the antigenicity of African swine fever virus p35 protein as diagnostic antigen

摘要:

为了筛选出酶联免疫吸附测定 (Enzyme linked immunosorbent assay，ELISA) 反应性最佳的非洲猪瘟病毒 (African swine fever virus，ASFV) 诊断抗原，通过建立ELISA方法，以杆状病毒昆虫细胞表达系统表达的ASFV p30蛋白诊断抗原为参照，首次探讨原核表达系统表达的ASFV p35蛋白作为诊断抗原的抗原性和潜力。免疫印迹和免疫荧光结果表明，获得了40 kDa的重组p35蛋白和30 kDa的p30蛋白，两种蛋白与ASFV阳性血清均具有较好的免疫反应原性。采用重组p30和p35蛋白作为诊断抗原分别建立ELISA方法，并验证其敏感性、稳定性以及与进口试剂盒的符合率。结果显示，尽管p35-ELISA方法的检测敏感性稍低于p30-ELISA方法，但其敏感性仍可达95.8%，且p35-ELISA方法和p30-ELISA方法的批内和批间变异系数均小于10%。p35-ELISA方法与进口试剂盒比较，符合率达97.2%。结果表明建立的p35-ELISA方法敏感性高且稳定性好，可应用于ASFV感染血清的检测。

关键词:非洲猪瘟病毒，p35蛋白，p30蛋白，抗原性，间接ELISA

Abstract:

In order to screen African swine fever virus (ASFV) diagnostic antigen with the best enzyme linked immunosorbent assay (ELISA) reactivity. By establishing the ELISA method, the diagnostic antigen of ASFV p30 protein expressed by baculovirus-insect cell expression system as reference, we explored the antigenic properties and diagnostic potential of ASFV p35 protein expressed by prokaryotic expression system as a diagnostic antigen. The results of Western blotting and immunofluorescence show that the molecular weight of the recombinant p35 protein and p30 protein obtained was 40 kDa and 30 kDa, respectively, and these two proteins had good immuno-reactivity with ASFV positive serum. Recombinant p30 and p35 proteins were used as diagnostic antigens to establish ELISA, and the sensitivity and repeatability of these methods were tested. The results show that although the detection sensitivity of the p30-ELISA established in this study was higher than that of the p35-ELISA, the sensitivity of p35-ELISA was 95.8%, and variations in intra- and inter-assay repeatability of the two methods were less than 10%. The coincidence rate between the p35-ELISA and the imported kit was 97.2%. Results show that p35-ELISA was sensitive and stable, and could detect specific antibodies against ASFV.

Key words:African swine fever virus, p35, p30, antigenicity, indirect ELISA