牛传染性鼻气管炎病毒gD蛋白表位鉴定  

Identification of an antigen epitope on the glycoprotein D of bovine rhinotracheitis virus

作　　者：周跃辉[1] 朱远茂[1] 任建乐[1] 严昊[1] 王雪枝[1] 马磊[1] 史鸿飞[1] 薛飞[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/大动物传染病研究室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2015年第11期871-874,898,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：哈尔滨市科技攻关计划项目(2012AA6BN020);公益性行业(农业)科研专项经费(201003060-04);国家科技支撑计划子课题(2012BAD12B03-3);兽医生物技术国家重点实验室基本科研业务专项经费(SKLVBP201424)

摘　　要：为鉴定牛传染性鼻气管炎病毒(IBRV)g D蛋白抗原表位,本研究利用p ET-30a原核表达系统表达、纯化了重组g D蛋白,采用超速离心纯化的IBRV免疫BALB/c小鼠,取脾淋巴细胞与SP2/0细胞进行融合,以重组g D蛋白作为包被抗原,通过间接ELISA筛选出一株稳定分泌抗g D蛋白的MAb杂交瘤细胞株(1G3)。间接免疫荧光结果表明:MAb与IBRV呈阳性反应,中和试验测定其腹水中和效价为1∶32。利用肽扫描技术原核表达GST融合短肽对MAb 1G3识别的抗原表位进行筛选鉴定,结果表明:该MAb识别的抗原表位为259EESKGYE265。蛋白序列分析表明该抗原表位在IBRV各分离株及牛疱疹病毒5型(Bo HV-5)中均保守。本研究为IBRV的糖蛋白g D的结构和免疫学特性及IBRV致病性的深入研究奠定了基础。To identify the epitope on glycoprotein D （gD） of infectious bovine rhinotracheitis virus （IBRV）, the SP2/0 cells were fused with spleen cells from BALB/c mice immunized with purified IBRV, and one hybridoma stably secreting monoclonal antibody （MAb） against gD of IBRV was identified by indirect ELISA coated with gD expressed in E.coli. The MAb was positively reacted with IBRV detected by indirect immunofluorescence assay. The titer of the MAb prepered in ascites was 1：32 by virus neutralization test. The antigen epitope was further identified with the MAb by the pepscan method using GST-fused peptides expressed in E.coli, and a linear epitope of 259EESKGYE265 was identified in the gD. Moreover, the deduced amino acid sequence alignment of IBRV gD sequences showed that the epitope was completely conserved among different isolates of IBRV and BoHV-5. The identification of the epitope would contribute to further study of the structure and immunogenicity of IBRV gD and the pathogenicity of IBRV.

关 键 词：牛传染性鼻气管炎病毒 gD蛋白 单克隆抗体 表位鉴定 

分 类 号：S852.65[农业科学—基础兽医学]