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

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。

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

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 monoclonal antibody against NSP8 protein was prepared by lymphocyte hybridoma technique.The monoclonal antibody belonged to IgG1 subtype with κ 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 ³¹SPQILKQLTKAFNIAKSDFEREASV⁵⁵ was identified by Western blotting with the monoclonal antibody.This monoclonal antibody and its epitope mapping provided a basis for further study of the function of the TGEV NSP8.