非洲猪瘟病毒p34蛋白单克隆抗体的制备及鉴定  

Preparation and Characterization of a Monoclonal Antibody Against p34 Protein of African Swine Fever Virus

作　　者：王晓 刘传霞 鲍苗菲 李雪雯 李婷婷 陈欣[1] 郑君[1] 翁长江[1] WANG Xiao;LIU Chuanxia;BAO Miaofei;LI Xuewen;LI Tingting;CHEN Xin;ZHENG Jun;WENG Changjiang(Division of Fundamental Immunology,State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,CAAS,Harbin 150069,China)

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基础免疫创新团队,哈尔滨150069

出　　处：《中国动物传染病学报》2024年第6期176-181,共6页Chinese Journal of Animal Infectious Diseases

基　　金：十四五国家重点研发计划项目(2021YFD1800100);国家自然科学基金项目(32172874)。

摘　　要：非洲猪瘟(ASF)是由非洲猪瘟病毒(ASFV)感染家猪或野猪而引起的一种高度接触性传染病。ASFV编码超过150种蛋白,其中大多数蛋白的功能有待鉴定。本研究旨在表达ASFV p34蛋白,并制备其单克隆抗体,为p34蛋白的功能研究提供材料。首先构建了重组原核表达质粒pET-21a-p34,将质粒转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导后,通过SDS-PAGE以及Western blot鉴定,结果显示,得到了可溶性表达的His-p34重组蛋白。将纯化的蛋白免疫小鼠,然后通过细胞融合以及ELISA筛选,获得了1株能够稳定分泌p34蛋白单克隆抗体的杂交瘤细胞株。经Western blot鉴定,该抗体能够特异性识别通过转染质粒过表达的外源p34蛋白以及ASFV感染猪肺泡巨噬细胞(PAMs)表达的内源p34蛋白;免疫荧光试验表明,外源转染以及内源表达的p34蛋白均能被特异性标记;免疫沉淀(IP)试验结果显示,该单克隆抗体能够特异性的结合HEK293T细胞中外源表达的p34蛋白以及ASFV感染PAMs中内源表达的p34蛋白。综上所述,本研究成功制备了p34蛋白的单克隆抗体,该抗体可以用于内源性p34蛋白的鉴定,为该蛋白的功能研究奠定了基础。African swine fever(ASF)is a highly contagious disease caused by African swine fever virus(ASFV)infection in domestic pigs and wild boars.ASFV encodes more than 150 proteins,of which most proteins remain to be characterized.This study aimed to express ASFV p34 protein and prepare monoclonal antibodies(mAbs)for the functional study.Firstly,the recombinant prokaryotic expression plasmid PET-21a-p34 was constructed and transformed into competent E.coli BL21(DE3)cells.The soluble His-p34 recombinant protein was expressed with IPTG induction and identified by SDS-PAGE and Western blot.The purified protein was used to immunize the mice.As a result,a hybridoma cell line capable of stably secreting the mAbs against p34 protein was obtained by cell fusion and ELISA screening.Western blot analysis showed that the mAb specifically recognized exogenous p34 protein overexpressed by transfected plasmid and endogenous p34 protein expressed by ASFV-infected porcine alveolar macrophages(PAMs).Immunofluorescence assay showed that both exogenous transfected and endogenous expressed p34 proteins were specifically labeled for visual reaction.Immunoprecipitation(IP)assay showed that the mAb specifically bound to exogenous p34 protein in HEK293T cells and endogenous p34 protein in ASFV-infected PAMs.In conclusion,the mAb to p34 protein was successfully prepared in this study,which could be used for the identification and functional study of endogenous p34 protein.

关 键 词：非洲猪瘟病毒 p34蛋白 原核表达 单克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]