H9N2亚型禽流感病毒多拷贝M2e蛋白单克隆抗体的制备

Preparation and Preliminary Application of Monoclonal Antibodies Against Multi-copy M2e Protein of H9N2 Subtype Avian Influenza Virus

为制备H9N2亚型禽流感病毒(AIV)多拷贝M2e蛋白单克隆抗体,试验将纯化的3M2e重组蛋白免疫BALB/c小鼠;取免疫小鼠的脾细胞与SP2/0细胞融合,采用间接ELISA筛选出分泌抗体的杂交瘤细胞株;将获得的杂交瘤细胞株腹腔注射BALB/c小鼠制备腹水;采用间接ELISA测定腹水效价,鼠源单克隆抗体亚类鉴定试剂盒检测单克隆抗体腹水,三株单克隆抗体重链亚型均为IgG1;Westernblot鉴定单克隆抗体的特异性;使用IFA鉴定3M2e蛋白在Sf9细胞的表达和检测H9N2亚型AIV在MDCK细胞的感染。结果显示:获得了3株分泌多拷贝M2e抗体的杂交瘤细胞株,分别命名为4C7-B11、4E10-A10和5A12-B5;获得腹水的效价均为1∶1000000;三株单克隆抗体重链亚型均为IgG1;Westernblot鉴定三株单克隆抗体均能与2M2e、3M2e和4M2e原核重组蛋白及在Sf9细胞表达的3M2e发生特异性反应;IFA鉴定结果也显示三株单克隆抗体均能检测3M2e蛋白在Sf9细胞的表达,并且能检测到H9N2亚型AIV在MDCK细胞上的感染,出现特异性的绿色荧光;三株单克隆抗体重链亚型均为IgG1。研究获得了H9N2亚型AIV多拷贝M2e单克隆抗体,可用于Westernblot和IFA鉴定,为靶向M2e蛋白H9N2亚型禽流感疫苗的研究提供特异性的检测工具。

To prepare monoclonal antibodies against the multi-copy M2e protein of H9N2 subtype avian influenza virus.BALB/c mice were immunized with the purified 3M2e recombinant protein;The spleen cells of the immunized mice were fused with SP2/0 cells,and then indirect ELISA method was used to screen hybridoma cell lines which can secrete specific antibodies;The ascites were obtained from BALB/c mice which were intraperitoneally injected with positive hybridoma cell lines;The titers of ascites were determined using indirect ELISA method,the subclass of monoclonal antibodies were determined using the mouse derived monoclonal antibody subclass identification kit,and the specificity of monoclonal antibodies was identified using Western blot.Three monoclonal antibodies were used to identify the expression of 3M2e protein in Sf9 cells and detect the infection of H9N2 subtype AIV in MDCK cells by IFA.The results showed that three hybridoma cell lines secreting multi-copy M2e antibodies were obtained,named 4C7-B11,4E10-A10 and 5A12-B5;The titers of ascites from the mice were all 1∶1000000;All three monoclonal antibody heavy chain subtypes were IgG1.Western blot test showed that three monoclonal antibodies could react specifically with 2M2e,3M2e,4M2e prokaryotic recombinant proteins;IFA test also showed that three monoclonal antibodies could detect 3M2e protein expressed in Sf9 cells and the infection of H9N2 subtype AIV in MDCK cells,specific green fluorescence was observed in Sf9 cells;IFA test also showed that three monoclonal antibodies could detect the infection of H9N2 subtype AIV in MDCK.The multi-copy M2e monoclonal antibodies of the H9N2 subtype AIV were obtained,which could be used for Western blot and IFA identification,providing a specific detection tool for the study of targeted M2e protein H9N2 subtype avian influenza vaccines.