非洲猪瘟CD2v胞外区基因片段的真核表达及其多克隆抗体的制备  

Eukaryotic expression the CD2v extracellular gene fragment of African swine fever virus and characterization of its polyclonal antibodies

作　　者：于浩洋 吴绍强[1] 王彩霞[1] Grzegorz WOZNIAKOWSKI 刘晓飞[1] 仇松寅[1] 李新实[1] 冯春燕[1] 林祥梅[1] YU Hao-yang;WU Shao-qiang;WANG Cai-xia;Grzegorz WOZNIAKOWSKI;LIU Xiao-fei;QIU Song-yin;LI Xin-shi;FENG Chun-yan;LIN Xiang-mei(The Institute of Animal Quarantine,Chinese Academy of Inspection and Quarantine,Beijing 100176,China;Department of Swine Diseases,National Veterinary Research Institute,Pulawy 24-100,Poland)

机构地区：[1]中国检验检疫科学研究院,北京100176 [2]波兰国家兽医研究所,普瓦维24-100

出　　处：《中国兽医科学》2021年第1期45-52,共8页Chinese Veterinary Science

基　　金：中国检验检疫科学研究院基本科研业务费项目(2020JK020);“十三五”国家重点研发计划项目(2016YFD0501100)。

摘　　要：本研究利用真核表达系统表达非洲猪瘟病毒的CD2v胞外区蛋白,研究其免疫特性,制备多克隆抗体并研究其性质,为研发特异性单抗奠定数据基础。以非洲猪瘟病毒Wuhan2019-1株(GenBank:MN393476.1)为模板,合成CD2v胞外区基因序列,并将其克隆至真核表达载体pCAGGS载体中,获得pCAGGS-CD2v胞外区重组质粒。经酶切和测序鉴定后,将重组质粒转染293F细胞,获得重组胞外CD2v蛋白(简称His-CD2v)。用Ni-NTA柱纯化His-CD2v,并以此为抗原免疫BALB/c小鼠制备多克隆抗体,利用ELISA和间接免疫荧光对多克隆抗体进行鉴定。结果显示,(1)His-CD2v融合蛋白在293F中以可溶形式表达,SDS结果显示,相对分子质量约为35~80 ku的弥散带;(2)融合表达的His-CD2v能与阳性血清反应,与对照不反应;(3)HisCD2v制备的多抗效价最高达1∶218700,且能与阳性血清竞争性结合CD2v抗原。上述结果表明,His-CD2v融合蛋白的表达及其多抗的成功制备,为进一步筛选CD2v特异性单抗及研制竞争ELISA试剂盒,为非洲猪瘟感染与免疫鉴别方法的建立奠定了坚实的基础。This study aimed to obtain the CD2 v protein of ASFV which was expressed by the eukaryotic expression system,and characterize the polyclonal antibodies of the recombination protein to lay the data foundation for the development of specific monoclonal antibody.To achieve this goal,the extracellular region of CD2 v gene sequence was synthesized and cloned into p CAGGS vector to obtain the recombinant plasmid p CAGGS-CD2 v.After identification by enzyme digestion and sequencing,the recombinant plasmid was transfected into 293 F cells to obtain the recombinant protein(His-CD2 v for short).Further,His-CD2 v was purified by NI-NTA column and used as antigen to immunize BALB/c mice to prepare polyclonal antibodies.Polyclonal antibodies were identified by ELISA and IFA.The results showed that:(1)His-CD2 v fusion protein was expressed in 293 F as a soluble protein,and its relative molecular weight was about 50 ku.(2)The highest titer of polyclonal antibody was 1∶218700.(3)The polyclonal antibody can react specifically with His-CD2 v fusion protein,and there is no cross reaction with control.The successful preparation of His-CD2 v fusion protein and its polyclonal antibodies laid the foundation for further screening of CD2 v-specific monoclonal antibodies,and also paved the way for establishing the discremination methods of infection and immunity for ASFV.

关 键 词：非洲猪瘟病毒 CD2v 真核表达 多克隆抗体 竞争ELISA 

分 类 号：S852.659.1[农业科学—基础兽医学]