猪传染性胃肠炎病毒NSP8蛋白单克隆抗体的制备及抗原表位的初步鉴定  

Preparation of Monoclonal Antibody and Preliminary Identification of Antigenic Epitope of NSP8 Protein in Porcine Transmissible Gastroenteritis Virus

作　　者：董慧[1] 张鑫[2] 陈建飞[2] 时洪艳[2] 石达[2] 常铁城 谷凤丽[2,4] 徐天[2] 王智琴[5] 刘明春[1] 冯力[2] 

机构地区：[1]沈阳农业大学畜牧兽医学院,沈阳110161 [2]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,猪传染病研究室,哈尔滨150001 [3]黑龙江八一农垦大学动物科技学院,大庆163319 [4]东北农业大学生命科学学院,哈尔滨150030 [5]首都医科大学基础医学院,北京100069

出　　处：《中国畜牧兽医》2015年第2期358-364,共7页China Animal Husbandry & Veterinary Medicine

基　　金：黑龙江省高等学校科技创新团队项目:猪病毒性腹泻疾病防治(2011TD001)

摘　　要：为鉴定猪传染性胃肠炎病毒(porcine transmissible gastroenteritis virus,TGEV)NSP8蛋白的抗原表位,本研究对GST-NSP8重组蛋白进行了原核表达,并用纯化后的重组蛋白免疫6周龄BALB/c小鼠,取免疫小鼠脾脏,采用常规杂交瘤细胞融合方法,经3次亚克隆后制备了1株稳定分泌抗NSP8蛋白的单克隆抗体杂交瘤细胞株。分泌的单克隆抗体亚类鉴定其重链为IgG1型,轻链为κ链;杂交瘤细胞培养上清的效价为1∶3 200。Western blotting试验结果表明该单克隆抗体能识别原核及真核表达的NSP8重组蛋白。利用截短表达的方法对NSP8蛋白进行抗原表位的鉴定,初步确定了单克隆抗体针对的抗原表位序列为31SPQILKQLTKAFNIAKSDFEREASV55。本研究制备的单克隆抗体及对抗原表位的鉴定,为TGEV NSP8蛋白相关功能的研究奠定了基础。To identify the epitope of porcine transmissible gastroenteritis virus （TGEV）, the NSP8 gene was cloned into the vector pGEX-6P-1 for expression in E. coli. BALB/c mice of 6 week-old were immunized with the recombinant NSP8 protein. Then the monoelonal antibody against NSP8 protein was prepared by lymphocyte hybridoma technique. The monoelonal antibody belonged to IgG1 subtype with k chain. The titers in cell culture medium of the hybidomas was 1 ： 3 200. Western blotting assay showed that the monoclonal antibody was able to recognize the recombinant NSP8 protein. Epitope on NSP8 protein was identified by truncated expression and the linear epitope of ^31SPQILKQLTKAFNIAKSDFEREASV^55 was identified by Western blotting with the monoclonal antibody. This monoelonal antibody and its epitope mapping provided a basis for further study of the function of the TGEV NSP8.

关 键 词：猪传染性胃肠炎病毒 NSP8基因 单克隆抗体 抗原表位 

分 类 号：S858.28[农业科学—临床兽医学]