非洲猪瘟病毒P30蛋白单克隆抗体的制备与应用

Preparation and Application of Monoclonal Antibodies against P30 Protein of African Swine Fever Virus

为研发非洲猪瘟病毒(ASFV)的免疫学诊断试剂,利用大肠杆菌表达系统表达重组ASFV P30蛋白,制备并鉴定P30蛋白单克隆抗体,建立检测ASFV的胶体金免疫层析法(GICA)。结果显示,成功构建了重组表达质粒pET28a-P30,实现了重组P30蛋白的可溶性表达,且该重组蛋白与ASFV阳性血清可发生特异性反应;制备了10株稳定分泌P30蛋白单克隆抗体的杂交瘤细胞株,腹水ELISA效价均高于1∶40000,与ASFV具有良好的反应性,且不与其他常见猪源病毒反应;利用2株单克隆抗体建立胶体金免疫层析法,可用于ASFV的检测。

In order to develop ASFV immunodiagnostic reagents,recombinant P30 protein of ASFV was prepared by Escherichia coli expression system.Monoclonal antibodies were prepared and identified to establish a gold immunochromatographic assay(GICA)for the detection of ASFV.The results showed that the recombinant expression plasmid pET28a-P30 was constructed successfully.Soluble recombinant P30 protein was expressed,and the protein could react with ASFV positive serum.Ten positive hybridoma cells stably secreting monoclonal antibodies against ASFV protein P30 were screened.The ascites ELISA titers of 10 hybridoma cells were higher than 1∶40000.These antibodies had no cross-reaction with other common swine viruses,and all of the 10 antibodies could react with ASFV.The obtained antibodies were used to establish a GICA,which could be used to detect ASFV.