非洲猪瘟病毒K205R蛋白的原核表达与单克隆抗体制备

Prokaryotic Expression of African Swine Fever K205R Protein and Preparation of Monoclonal Antibody

为研发非洲猪瘟病毒的免疫检测试剂提供素材,制备非洲猪瘟病毒K205R蛋白的单克隆抗体。采用PCR方法扩增K205R基因,构建重组表达质粒pET-30a-K205R,将重组质粒pET-30a-K205R转化到大肠埃希氏菌BL21(DE3)中并通过IPTG诱导表达ASFV K205R蛋白。将纯化出的蛋白乳化后免疫小鼠,采用杂交瘤技术制备能稳定分泌单克隆抗体的杂交瘤细胞。将筛选出来的单克隆细胞注射至小鼠腹腔中,待小鼠腹部膨大时收集腹水,通过ELISA方法鉴定腹水效价、Western blot和IFA鉴定单克隆抗体特异性及反应原性,并对单克隆抗体亚型进行鉴定。经SDS-PAGE鉴定,重组ASFV K205R蛋白成功表达。Western blot分析显示,His标签抗体能够特异性识别重组表达的ASFV K205R蛋白,表明其具有良好的特异性以及反应原性。通过细胞融合和细胞筛选得到1株能稳定分泌抗ASFV K205R蛋白单克隆抗体的杂交瘤细胞,将其命名为1C3G6。ELISA检测腹水中抗体效价为1∶100000。Western blot和IFA鉴定结果显示单克隆抗体1C3G6与K205R蛋白反应性良好。通过亚型鉴定,单克隆抗体1C3G6的重链为IgG2b类,轻链为Kappa型。经Western blot和IFA鉴定,筛选得到的1C3G6单克隆抗体可以特异性识别原核系统和真核系统表达的K205R蛋白,说明该抗体具有良好的特异性。

In order to prepare the monoclonal antibody of African swine fever virus K205R protein and provide materials for the development of African swine fever virus immunodetection reagents.In this study,The PCR was used to amplify the K205R gene,and the recombinant expression plasmid pET-30a-K205R was constructed.The recombinant plasmid pET-30a-K205R was transformed into Escherichia coli BL21(DE3)and induced to express ASFV K205R protein by IPTG.The purified protein was emulsified and immunized to mice,preparation of hybridoma cells capable stably secreting monoclonal antibodies using hybridoma technology.The screened monoclonal cells were injected into the abdominal cavity of mice,and the ascites was collected when the abdomen of the mice was distended.The recombinant ASFV K205R protein was successfully expressed and identified by SDS-PAGE.Western blot analysis showed that the His-tag antibody could specifically recognize the recombinantly expressed ASFV K205R protein,indicating that it has good specificity and reactogenicity.A monoclonal antibody hybridoma cell line that can stably secrete anti-ASFV K205R protein was obtained by cell fusion and cell screening,which was named 1C3G6.The antibody titer of the ascites fluid detected by ELISA was 1∶100000.The results of Western blot and IFA identification showed that the monoclonal antibody 1C3G6 had good reactivity and specificity with K205R protein.By isotype identification,the heavy chain of monoclonal antibody 1C3G6 is IgG2b,and the light chain is Kappa.The 1C3G6 monoclonal antibody screened can specifically recognize the K205R protein expressed in prokaryotic systems by Western blot and IFA identification,indicating that the antibody has good specificity.