Development of a recombinant pB602L-based indirect ELISA assay for detecting antibodies against African swine fever virus in pigs

doi:10.1016/S2095-3119(21)63767-X

摘要/Abstract

摘要：

非洲猪瘟 (ASF) 是家猪和野猪的一种毁灭性疾病。目前没有有效的商品化疫苗，疾病的控制主要依赖于对感染猪的监测和早期检测。ASF是由非洲猪瘟病毒 (ASFV) 引起的疾病。以前的血清学检测方法（如 ELISA）主要基于ASFV的重组结构蛋白，包括 p72、p54和p30等。然而，猪感染 ASFV后产生的针对此类蛋白的抗体不能为其提供有效的免疫保护。因此，仍然需要可用于临床诊断和评估疫苗免疫猪体液免疫反应的新血清学检测方法。在此项研究中，我们表达并纯化了重组pB602L蛋白。然后将纯化的pB602L蛋白作为抗原进行间接 ELISA 检测。该方法对猪瘟病毒、伪狂犬病病毒、猪细小病毒等15种最常见的猪病原的抗血清无交叉反应。然后使用该方法与商品化ELISA试剂盒同时对60头猪的血清样本进行检测，其中包括数量未知的抗ASFV血清，两种方法检测的符合率为95%。此外，使用基于pB602L蛋白的iELISA对7基因缺失的ASFV株（HLJ/18-7GD）免疫猪体内的抗体水平进行检测。结果表明，接种10 天以后，在所有接种猪体内都检测到抗体水平持续增加。我们的结果表明，这种基于 pB602L的间接ELISA检测可用于ASFV的临床诊断。

Abstract: African swine fever (ASF), caused by the African swine fever virus (ASFV), is a devastating disease of domestic and wild pigs. There is no effective vaccine, and the control of the disease relies mainly on surveillance and early detection of infected pigs. Previously, serological assays, such as ELISA, have been developed mainly based on recombinant structural viral proteins of ASFV, including p72, p54, and p30. However, the antibodies against these proteins do not provide efficient protection against ASFV infection in pigs. Therefore, new serological assays that can be applied for clinical diagnosis and evaluating serological immune response in vaccinated pigs are still required. In this study, we expressed and purified a recombinant pB602L protein. The purified pB602L protein was then used as an antigen to develop an indirect ELISA assay. This assay has no cross-reaction with the anti-sera against the 15 most common pig pathogens in China, such as classical swine fever virus, pseudorabies virus, and porcine parvovirus. This assay and a commercial ELISA kit were then used to detect 60 field pig serum samples, including an unknown number of anti-ASFV sera. The coincidence of the two assays was 95%. Furthermore, the pB602L-based ELISA was employed to test the antibody responses to the seven-gene-deleted ASFV strain HLJ/18-7GD in pigs. The results showed that the antibody levels in all vaccinated pigs, starting from the 10th day post-inoculation, have increased continuously during the observation period of 45 days. Our results indicate that this pB602L-based indirect ELISA assay can be employed potentially in the field of ASFV diagnosis.

Key words: African swine fever virus , pB602L , recombinant protein , indirect ELISA assay , pig

. [J]. Journal of Integrative Agriculture, 2022, 21(3): 819-825.

WANG Peng-fei, WANG Ming, SHI Zhi-bin, SUN Zhen-zhao, WEI Li-li, LIU Zai-si, WANG Shi-da, HE Xi-jun, WANG Jing-fei. Development of a recombinant pB602L-based indirect ELISA assay for detecting antibodies against African swine fever virus in pigs[J]. Journal of Integrative Agriculture, 2022, 21(3): 819-825.

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