猪流行性腹泻病毒N蛋白的原核表达及其单克隆抗体的制备  

PROKARYOTIC EXPRESSION OF PORCINE EPIDEMIC DIARRHEA VIRUS N PROTEIN AND PREPARATION OF MONOCLONAL ANTIBODIES

作　　者：李任峰[1] 田香勤[2] 韩笑 王晓斐 姜金庆[1] 夏小静[1] 王异民 王自良[1] LI Ren-feng;TIAN Xiang-qin;HAN Xiao;WANG Xiao-fei;JIANG Jin-qing;XIA Xiao-jing;WANG Yi-min;WANG Zi-liang(College of Animal Science and Veterinary Medicine,Henan Institute of Science and Technology,Xinxiang 453003,China;Key Laboratory of Henan Province for Medical Tissue Regeneration,Xinxiang Medical University,Xinxiang 453003,China)

机构地区：[1]河南科技学院动物科技学院,新乡453003 [2]新乡医学院河南省医用组织再生重点实验室,新乡453003

出　　处：《中国动物传染病学报》2020年第4期52-57,共6页Chinese Journal of Animal Infectious Diseases

基　　金：河南省科技攻关项目(192102110190);河南省科技攻关项目(202102110255);河南省高校科技创新团队计划(20IRTSTHN025)。

摘　　要：本研究旨在采用原核表达系统表达猪流行性腹泻病毒(PEDV)的核衣壳蛋白(N),并进一步制备其单克隆抗体。将构建重组原核表达质粒pET28a-N转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导表达重组蛋白,并采用Ni-NTA亲和层析和分子筛进行纯化。将纯化后的重组N蛋白免疫BALB/c小鼠,取小鼠脾脏B淋巴细胞与SP2/0瘤细胞融合,采用间接ELISA方法筛选阳性杂交瘤细胞株,并对其分泌抗体的特性进行了分析。结果显示:重组质粒pET28a-N在E.coli BL21中高效表达,筛选获得了2株稳定分泌抗N蛋白单克隆抗体的杂交瘤细胞株1A3和4F6,其分泌的抗体均为IgG1亚型,腹水效价分别为5.12×10^5、1.28×10^5。本研究为进一步研究N蛋白的功能以及建立PEDV免疫学诊断方法奠定了基础。In order to obtain nucleocapsid(N)protein of porcine epidemic diarrhea virus(PEDV)and prepare monoclonal antibodies(McAb),the recombinant plasmid pET28a-N was constructed and expressed in BL21(DE3)competent cells after induced with IPTG.Then,the recombinant N protein was purified by Ni-NTA affinity and gel filtration chromatography and used to immunize BALB/c mice.The splenocytes from the immunized mice were fused with SP2/0 myeloma cells and the hybridomas were screen for secretion of MAbs by indirect ELISA.As a result,2 hybridoma cell lines(1A3 and 4F6)that stably produced anti-N protein antibodies were obtained and further analyzed.The antibody subtypes of both McAbs were IgG1.The antibody titers of 1A3 and 4F6 in ascites were 5.12×10^5 and 1.28×10^5 as determined in indirect ELISA,respectively.This study laid an important foundation for the functional research of N protein and development of diagnostic kits of PEDV.

关 键 词：猪流行性腹泻病毒 N蛋白 原核表达 单克隆抗体 

分 类 号：S852.659.6[农业科学—基础兽医学]