非洲猪瘟病毒pE120R蛋白单克隆抗体的制备  

Preparation of Monoclonal Antibody of African Swine Fever Virus pE120R

作　　者：刘传霞 王晓[1] 李雪雯 鲍苗菲 李婷婷 陈欣[1] 翁长江[1] 郑君[1] LIU Chuanxia;WANG Xiao;LI Xuewen;BAO Miaofei;LI Tingting;CHEN Xin;WENG Changjiang;ZHENG Jun(Division of Fundamental Immunology,National African Swine Fever Para-Reference Laboratory,State Key Laboratory for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所、动物疫病预防控制国家重点实验室国家非洲猪瘟专业实验室基础免疫创新团队,哈尔滨150069

出　　处：《畜牧兽医学报》2024年第1期388-394,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：“十四五”国家重点研发计划项目(2021YFD1800100);国家自然科学基金(32172874)。

摘　　要：本研究旨在表达非洲猪瘟病毒(ASFV)衣壳蛋白pE120R,并制备单克隆抗体,为其功能研究提供材料。以pCAGGS-Flag-pE120R为模板扩增pE120R基因片段,构建原核表达质粒pGEX-6p1-pE120R。将其转化BL21(DE3)感受态细胞后经IPTG诱导表达为可溶性pE120R蛋白,采用GST Beads纯化pE120R蛋白,免疫BALB/c小鼠,然后取其脾细胞与SP2/0细胞进行细胞融合,采用间接ELISA方法筛选获得了1株能够稳定分泌pE120R单克隆抗体的杂交瘤细胞株。结果表明,成功构建了原核表达质粒pGEX-6p1-pE120R,并获得较高纯度的pE120R蛋白。Western blot和间接免疫荧光(IFA)试验结果显示,pE120R单抗能特异性识别HEK293T细胞转染质粒表达的Flag-pE120R蛋白和肺泡巨噬细胞(PAMs)感染ASFV后表达的pE120R蛋白。该单抗的结合位点在30~60aa区域,并且该单抗亚类鉴定重链为IgG2a。本研究成功表达并纯化了可溶性的pE120R蛋白;制备了抗pE120R的单抗隆抗体,该抗体具有良好的特异性,为深入探讨ASFV pE120R蛋白的生物学功能奠定了基础。This study aimed to express the African swine fever virus(ASFV)capsid protein pE120R and prepare the monoclonal antibody(mAb)for the further study of its function.The pE120R gene was amplified from pCAGGS-Flag-pE120R and the prokaryotic expression plasmid pGEX-6p1-pE120R was constructed.After transforming into BL21(DE3)competent cells,it was induced to express soluble pE120R protein by IPTG.pE120R protein was purified by GST Beads and immunized BALB/c mice,the spleen cells of the immunized mice were collected and perform cell fusion with SP2/0 cell,thus a hybridoma cell line secreting pE120R monoclonal antibody was obtained by indirect ELISA.The results showed that the prokaryotic expression plasmid pGEX-6p1-pE120R was successfully constructed and the high purity pE120R protein was obtained.The results of Western blot and indirect immunofluorescence assay(IFA)showed that pE120R mAb could specifically recognize Flag-pE120R protein expressed by HEK293T cells transfected with plasmid and pE120R protein expressed by ASFV infected alveolar macrophage cells(PAMs).The binding site of this mAb is in the 30-60 aa region,and the mAb subclass identifies the heavy chain as IgG2a.In this study,the soluble pE120R protein was successfully expressed and purified;the monoantibody against pE120R was prepared,which has good specificity and laid a foundation for exploring the biological function of ASFV pE120R protein.

关 键 词：非洲猪瘟病毒 pE120R蛋白 原核表达 单抗隆抗体 

分 类 号：S852.651[农业科学—基础兽医学]