H9亚型禽流感病毒基质蛋白M1单克隆抗体的制备与鉴定  

PREPARATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST M1 PROTEIN OF H9N2 AVIAN INFLUENZA VIRUS

作　　者：戴晓光[1,2] 滕巧泱[2] 黄海碧[2] 王朝霞[2] 张树梅[2] 张旭[2] 徐大伟[2] 申之义[1] 李泽君[2] 

机构地区：[1]内蒙古农业大学兽医学院,呼和浩特010018 [2]中国农业科学院上海兽医研究所农业部动物寄生虫学重点开放实验室,上海200241

出　　处：《中国动物传染病学报》2010年第3期8-12,共5页Chinese Journal of Animal Infectious Diseases

基　　金：上海市浦江人才计划(09PJ1411900)

摘　　要：本研究用纯化的H9N2亚型禽流感病毒免疫BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合。对杂交瘤细胞及时筛选,阳性孔经3次有限稀释法克隆,成功获得3株能稳定传代并分泌抗H9亚型禽流感病毒基质蛋白M1单克隆抗体的杂交瘤细胞:3G8、2F6、5F2。间接ELISA方法检测,3株单克隆抗体的腹水间接ELISA效价达106以上。构建了真核表达载体pCAGGS-M1并转染于MDCK细胞,以用于腹水的Western blot和间接免疫荧光鉴定。间接免疫荧光结果表明,3株单克隆抗体皆与真核表达蛋白M1反应。这些单克隆抗体的制备为后期研究M1蛋白在流感病毒复制与出芽过程中的重要生物学功能奠定了基础。BALB/c mice were immunized with the H9N2 subtype avian influenza virus and their spleen cells were fused with mouse myeloma cells（SP2/0）.Three stable hybridoma cell lines（3G8,2F6,5F2） secreting monoclonal antibodies（mAbs） against matrix protein M1 were identified after three rounds of limited dilutions.The titers of ascetic fluids of these three mAbs were up to 106 by indirect ELISA.In order to test the mAbs in Western blot and indirect inmmunofluorescene assay（IFA）,the recombinant eukaryotic expression vector pCAGGS-M1 was constructed and transfected into MDCK cells.The results of IFA demonstrated that MAbs could specifically bind to eukaryotic M1 protein.However,these MAbs could not react with M1 protein in Western blots,suggesting they might be specific for structural epitopes of M1.These MAbs can be used to analyze biological function of M1 protein in the process of virus replication and budding.

关 键 词：H9亚型禽流感病毒 基质蛋白M1 单克隆抗体 间接免疫荧光 蛋白免疫印迹法 

分 类 号：S852.659.5[农业科学—基础兽医学] Q78[农业科学—兽医学]