猪瘟病毒E2蛋白单克隆抗体的制备及抗原表位的鉴定  

Preparation of monoclonal antibody to swine fever virus E2 protein and identification of antigenic epitopes

作　　者：林琪虹 李长尧 张朝霞[2,3] 宋厚辉 翁长江 LIN Qi-hong;LI Chang-yao;ZHANG Zhao-xia;SONG Hou-hui;WENG Chang-jiang(College of Animal Science and Technology-School of Animal Medicine,Zhejiang Agriculture and Forestry University,Hangzhou 311300,China;National Key Laboratory of Animal Disease Prevention and Control,Harbin Institute of Veterinary Medicine,Chinese Academy of Agricultural Sciences/Basic Immunization Innovation Team,Harbin 150069,China;Key Laboratory of Veterinary Immunization,Heilongjiang Province,Harbin 150069,China)

机构地区：[1]浙江农林大学动物科技学院动物医学院,浙江杭州311300 [2]中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室/基础免疫创新团队,黑龙江哈尔滨150069 [3]黑龙江省兽医免疫学重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2025年第2期179-184,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省自然科学基金资助项目(C2016061)。

摘　　要：为制备猪瘟病毒(CSFV)鼠源E2蛋白单克隆抗体(MAb)并鉴定其抗原表位,本研究通过昆虫细胞杆状病毒表达系统表达并纯化了重组E2蛋白(rE2),将其免疫BALB/c小鼠后通过间接ELISA方法筛选到1株能够稳定分泌E2蛋白MAb的杂交瘤细胞株3D5。经MAb亚类鉴定试剂盒检测结果显示3D5 MAb为IgM型。采用western blot检测制备的3D5 MAb与293T细胞中过表达的E2蛋白及CSFV感染猪肾细胞(PK-15细胞)后(感染后不同时间)天然表达E2蛋白的反应性;采用间接免疫荧光试验(IFA)检测3D5 MAb与PK-15细胞中天然表达E2蛋白的反应性。Western blot结果显示,过表达E2蛋白的293T细胞和感染CSFV的PK-15细胞中(感染后12 h)均出现了40 ku左右的特异性条带。IFA结果显示,感染CSFV的PK-15细胞中出现红色荧光。以上结果表明,制备的3D5 MAb均能够与过表达的和天然表达的E2蛋白有较强的反应性。利用一系列表达截短的重组E2片段,通过western blot鉴定3D5 MAb识别的抗原表位,结果显示,E2蛋白氨基酸序列中的157REKPFPHRMD167为3D5 MAb的抗原表位。本研究首次制备了CSFV E2蛋白的MAb,并对其进行了表位鉴定,为进一步深入研究CSFV E2蛋白的结构、功能、抗原表位的筛选及新疫苗的研制提供参考依据。In this study,the recombinant E2 protein(rE2)of classical swine fever virus(CSFV)was expressed and purified by an insect cell baculovirus expression system to prepare amurine monoclonal antibody(MAb)against the E2 protein.After the recombinant E2 protein(rE2)was expressed and purified by an insect cell baculovirus expression system,rE2 was immunized into BALB/c mice and a hybridoma cell line 3D5 that stably secrete the E2 protein MAb was generated,which was screened by indirect ELISA.The results of the subclass identification showed that the 3D5 MAb was of the IgM type.The reactivity of the prepared 3D5 MAb with overexpressed E2 protein in 293T cells and naturally expressed E2 protein in CSFV-infected porcine kidney cells(PK-15 cells)(at different times after infection)was detected by western blot,and the reactivity of 3D5 MAb with naturally expressed E2 protein in PK-15 cells was detected by indirect immunofluorescence assay(IFA).Western blot results showed that 293T cells overexpressing E2 protein showed a specific band of approximately 40ku.A specific band of about 40ku appeared in PK-15 cells infected with CSFV(12 hours after infection).IFA results showed that red fluorescence appeared in PK-15 cells infected with CSFV.The above results indicate that the prepared 3D5 MAb was able to specifically and strongly react with both overexpressed and naturally expressed E2 proteins.To identify the epitope recognized by 3D5 MAb,a series of partially overlapping recombinant E2 fragments were designed,expressed and the reactivity of each E2 fragment to 3D5 MAb was analysed by western blot.The results showed that the antigenic epitope of 3D5 MAb lays between 157REKPFPHRMD167 in the amino acid sequence of the E2 protein.The present study successfully prepared a MAb against E2 protein of CSFV for the first time and its epitope was identified,which provides a basis for further indepth research on the structure,function,antigenic epitope screening and development of new vaccines for CSFV E2 protein.

关 键 词：猪瘟病毒 E2蛋白 单克隆抗体 抗原表位 

分 类 号：S852.65[农业科学—基础兽医学]