口蹄疫病毒非结构蛋白2C主要表位区的原核表达及其单克隆抗体的制备  

Preparation of McAb against non-structural protein of FMDV using a truncated 2C protein expressed in E.coli

作　　者：田美娜[1] 李永亮[1] 付元芳[1] 卢曾军[1] 袁明 吴磊[1] 刘在新[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室／农业部畜禽病毒学重点实验室／口蹄疫国家参考实验室,甘肃兰州730046 [2]河北畜牧良种工作站,河北石家庄050061

出　　处：《中国预防兽医学报》2009年第1期69-73,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研究973项目(2005CB523201)

摘　　要：本研究制备并鉴定了口蹄疫病毒非结构蛋白2C的单克隆抗体(mAb)。以纯化的原核表达2C蛋白免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞融合,所获得的杂交瘤细胞经间接ELISA法筛选,有限稀释法克隆,直至所克隆的细胞能够100%的分泌抗体,用Dot-ELISA以及IFA对mAbs的特异性等进行鉴定。共获得4株抗重组蛋白2C的mAbs,其亚类鉴定结果3株为IgG1,1株为IgG2b,叠加ELISA初步判定4株单抗所识别表位相同或相近。Dot-ELISA显示这些mAbs与重组2C蛋白和(2C'3AB)蛋白以及口蹄疫病毒O/China99感染的BHK-21细胞中的2C抗原均可特异性结合,IFA结果显示所得单抗能与FMDV感染细胞中的2C蛋白特异性结合。本研究获得的针对2C蛋白的特异性mAb,为进一步研究2C蛋白的结构和功能以及诊断方法奠定了基础。To prepare monoclonal antibodies specific to the non-structure protein of FMDV, BALB/c mice were immunized with 2C protein expressed from E.coli and splenocytes were fused with myeloma cells SP2/0 to produce hybridoma. Specific antibody was screened by indirect ELISA and positive hybridoma cells were cloned by limiting dilution assay. Immnofluorescence assay （IFA） was applied to identify antibodies. Four hybridoma cell lines steadily secreting antibodies against non- structure protein of FMDV were obtained, one was IgG2b subtype and the others were IgG1 subtype, and all have identical recognition sites. These antibodies bound specifically to both natural 2C protein and recombinant non-structure protein of FMDV. This work provided a foundation for establishing early diagnosis and stadying the structure and function of FMDV.

关 键 词：口蹄疫病毒 非结构蛋白2C 大肠杆菌表达 单克隆抗体 

分 类 号：S852.3[农业科学—基础兽医学]