抗东方马脑炎病毒结构蛋白E2单克隆抗体的制备及其抗原表位的鉴定  

Preparation of monoclonal antibodies against eastern equine encephalitis virus E2 protein and the B-cell epitopes identification

作　　者：赵晶[1] 孙恩成[1] 刘霓红[1] 杨涛[1] 杨银辉[2] 耿宏伟[1] 秦永丽[1] 王凌凤[1] 徐青元[1] 吴东来[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/农业部兽医公共卫生重点开放实验室,黑龙江哈尔滨150001 [2]军事医学科学院微生物流行病研究所病原微生物/生物安全国家重点实验室,北京100071

出　　处：《中国预防兽医学报》2011年第11期893-897,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家863项目(2011AA10A212);黑龙江省自然科学基因项目(ZJN-0602-01)

摘　　要：为制备抗东方马脑炎病毒(EEEV)结构蛋白E2的单克隆抗体(MAb)并鉴定其抗原表位,本研究以Bac-to-Bac真核表达系统表达EEEV E2蛋白,纯化后作为免疫原免疫BALB/c小鼠,取其脾淋巴细胞与小鼠骨髓瘤细胞SP2/0进行融合。以原核表达载体pET-30a表达并纯化的EEEV E2蛋白作为包被抗原建立间接ELISA方法筛选杂交瘤细胞,获得4株稳定分泌抗EEEV E2蛋白MAbs的杂交瘤细胞株,分别命名为6F3、6F11、7C11、8B11。Western blot与间接免疫荧光试验结果表明,获得的4株MAbs均与EEEV呈阳性反应,而与西方马脑炎病毒、乙型脑炎病毒以及登革热病毒1型～4型呈阴性反应。利用部分重叠的原核表达的短肽对E2蛋白抗原表位进行鉴定,初步确定MAb 6F11、7C11和8B11识别的抗原表位均为E-33(321EGLEYTWGNHPPKRVW336),而MAb 6F3无短肽与其反应,推测可能为构象表位。本研究结果为建立EEEV型特异性检测方法、研究E2蛋白结构功能及该病的进一步防制奠定了基础。For preparation of monoclonal antibodies（MAb） against eastern equine encephalitis virus（EEEV） structural glycoprotein E2 and identification of the B-cell epitopes,BALB/c mice were immunized with protein E2 expressed in Bac-to-Bac eukaryotic expression system.Splenocytes of the BALB/c mice were fused with myeloma cell SP2/0 for producing hybridoma.Four hybridomas stably secreting MAbs against the protein E2 were developed,designated 6F3,6F11,7C11 and 8B11,and determined by indirect ELISA,coating with purified EEEV E2 expressed in E.coli.The western blot and IFA demonstrated that the MAbs were able to react positively with EEEV,whereas no cross reaction was found for western equine encephalitis virus,Japanese encephalitis virus and Dengue virus serotype 1 to 4.Further epitope mapping of EEEV E2 protein with a set of shortpeptides showed that MAb 6F11,7C11 and 8B11 recognized the linear epitope at E-33（321EGLEYTWGNHPPKRVW336）,but not for MAb 6F3 which might be a conformation epitope.The preparation of MAbs and identification of E2 epitopes might have potential applications in EEEV serotype-specific detection method establishment,and structural and functional study of EEEV E2.

关 键 词：东方马脑炎病毒 E2蛋白 原核表达 真核表达 单克隆抗体 抗原表位 

分 类 号：S852.65[农业科学—基础兽医学]