非洲猪瘟病毒p22蛋白的表达及单克隆抗体制备

Expression and Monoclonal Antibody Preparation of the p22 Protein of African Swine Fever Virus

为研发非洲猪瘟病毒的免疫诊断学试剂,利用Bac-to-Bac杆状病毒表达系统表达非洲猪瘟病毒p22蛋白,制备抗p22蛋白单克隆抗体并鉴定其与非洲猪瘟病毒的反应性。结果显示,成功制备重组杆状病毒AcNPV-p22,表达得到大小约22 ku可溶性的重组p22蛋白。Western blot鉴定结果表明,重组p22蛋白可以与非洲猪瘟病毒阳性血清发生特异性反应。筛选到能够稳定分泌抗非洲猪瘟病毒p22蛋白单克隆抗体的杂交瘤细胞株(3D2、4A7),腹水ELISA效价均高于1∶128 000;单克隆抗体亚类分型鉴定结果表明,重链均为IgG1,轻链均为κ;间接免疫荧光试验(IFA)结果表明,制备的单克隆抗体能与非洲猪瘟病毒发生特异性反应。综上,利用杆状病毒-昆虫表达系统成功表达了可溶性的非洲猪瘟病毒重组p22蛋白,并制备抗p22蛋白单克隆抗体。

In order to develop an immunodiagnostic reagent for African swine fever virus,the Bac-to-Bac baculovirus expression system was used to express the p22 protein of African swine fever virus,the anti-p22 protein monoclonal antibody was prepared and its reactogenicity with African swine fever virus was identified. The results showed that the recombinant baculovirus AcNPV-p22 was successfully obtained,and the soluble recombinant protein of p22 with the size of about 22 ku was expressed. Western blot analysis confirmed that the protein had a good reactivity with African swine fever virus positive serum. Positive hybridoma cells(3 D2,4 A7)stably secreting monoclonal antibodies against African swine fever virus p22 protein were screened. The ascites ELISA titers of the two hybridoma cells were higher than 1∶128 000. Monoclonal antibodies isotype assay showed that the heavy chain was IgG1,and the light chain was κ. The IFA results showed that the prepared monoclonal antibody could specifically react with African swine fever virus. In summary,this study successfully expressed African swine fever virus p22 protein by baculovirus-insect expression system and the monoclonal antibodies against African swine fever virus p22 protein were successfully prepared.