裂谷热病毒N蛋白的原核表达及其单克隆抗体的制备  

Prokaryotic Expression and Preparation of Monoclonal Antibodies for N Protein of Rift Valley Fever Virus

作　　者：傅欣玲 张聪 张纹纹[1] 李文良[1] 杨蕾蕾[1] 毛立[1] 李基棕[1] 江杰元[1] FU Xinling;ZHANG Cong;ZHANG Wenwen;LI Wenliang;YANG Leilei;MAO Li;LI Jizong;JIANG Jieyuan(Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agriculture and Rural Affairs,Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology,Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;College of Wterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]江苏省农业科学院兽医研究所农业农村部兽用生物制品工程技术重点实验室江苏省食品质量安全重点实验室-省部共建国家重点实验室培育基地,南京210014 [2]南京农业大学动物医学院,南京210095

出　　处：《中国动物传染病学报》2021年第3期87-92,共6页Chinese Journal of Animal Infectious Diseases

基　　金：“十三五”国家重点研发计划(2016YFD0500908);江苏省农业科技自主创新资金项目(CX(18)2003)。

摘　　要：裂谷热(RVF)是由裂谷热病毒(RVFV)引起的一种人兽共患传染病,主要由蚊媒传播。该病主要感染反刍动物,可引起流产和新生胎儿死亡,人类对该病也易感,严重者可导致死亡。对于无RVF的国家,建立相应的病原学与血清学检测方法对于防止该病传入至关重要。为了建立基于病毒N蛋白的诊断技术,本研究构建了裂谷热病毒N基因原核表达质粒pET-28a-N,转化BL21(DE3),IPTG诱导表达,通过SDS-PAGE和Western blot检测蛋白的表达,重组蛋白分子量约为29 kDa,主要以上清液形式存在。大量表达并纯化重组蛋白,用纯化后的重组蛋白免疫BALB/c小鼠,制备单克隆抗体,并对其进行鉴定。采用间接ELISA和ID Vet公司阻断ELISA试剂盒检测免疫小鼠抗体效价及特异性,并用Western blot检测多克隆抗体反应性。结果表明,免疫后的抗体效价迅速升高,最终可达51200以上;阻断ELISA和Western blot检测结果表明,制备的多克隆抗体具有良好的反应性与特异性。通过3次亚克隆最终获得2株分泌单克隆抗体的杂交瘤细胞株,分泌的单克隆抗体均可与重组蛋白反应,重链均为IgG2a型,轻链为κ型。本研究为RVF诊断方法的建立奠定了基础。Rift valley fever(RVF)is a zoonotic infectious disease caused by Rift valley fever virus(RVFV),which was mainly transmitted by mosquitoes.The disease mainly infects ruminants,causing abortion and death of newborn babies.Humans are also susceptible to RVFV infection and may be dead under the serious conditions.For countries without RVF,the development of appropriate etiological and serological detection methods is essential to prevent the spread of the disease.In this study,the prokaryotic expression plasmid pET-28a-N containing RVFV N gene was constructed and transformed into BL21(DE3).The recombinant protein expression was induced with IPTG and detected by SDS-PAGE and Western blot.The results showed that the recombinant protein of a molecular weight at about 29 kDa was expressed and released mainly into the supernatant.The recombinant protein was purified by His purification columns.BALB/c mice were immunized with the purified protein to prepare monoclonal antibodies(mAbs).The mouse serum samples were collected and tested by indirect ELISA,ID Vet ELISA kit and Western blot.The results showed that the antibody titers increased rapidly after immunization(titers>51200)and showed good reactivity and specificity.After three rounds of sub-cloning,two monoclonal cell lines secreting mAbs specific to N protein were obtained.For these two mAbs,their heavy chain types were IgG2a and light chain types wereκ.These results laid a foundation for the development of diagnostic method of RVF.

关 键 词：裂谷热病毒 N蛋白 原核表达 单克隆抗体 

分 类 号：S852.659.5[农业科学—基础兽医学]