非洲猪瘟病毒CP312R蛋白的真核表达及单克隆抗体制备
Eukaryotic expression and monoclonal antibody preparation of CP312R recombi-nant protein of African swine fever virus
颜世君 1赵少若 2郝丽影 2白露露 1王同燕 1邓均华 2田克恭3
作者信息
- 1. 国家兽用药品工程技术研究中心,河南洛阳 471000;普莱柯生物工程股份有限公司,河南洛阳 471000
- 2. 洛阳普泰生物技术有限公司,河南洛阳 471000
- 3. 国家兽用药品工程技术研究中心,河南洛阳 471000;普莱柯生物工程股份有限公司,河南洛阳 471000;洛阳普泰生物技术有限公司,河南洛阳 471000
- 折叠
摘要
为制备非洲猪瘟病毒(African swine fever virus,ASFV)CP312R蛋白的单克隆抗体,以真核表达ASFV的CP312R蛋白为免疫原免疫BALB/c小鼠制备单克隆抗体,并通过间接免疫荧光法(IFA)对获得的单克隆抗体进行反应性鉴定.结果利用杆状病毒表达系统(baculovirus expression system,BES)构建获得重组转移载体pOET3-CP312R,与杆状病毒基因组flashBAC™ ULTRA共转染Sf9昆虫细胞获得重组杆状病毒AcMNPV-CP312R,且该病毒以胞内可溶形式表达出重组ASFV CP312R蛋白质.通过蛋白质印迹法鉴定显示重组CP312R蛋白可以与AS-FV 阳性血清发生特异性反应.此外,筛选获得2株针对重组CP312R蛋白的单克隆抗体(McAb),间接ELISA方法鉴定抗体滴度不低于1∶1 024 000.IFA试验检测表明,单克隆抗体与非洲猪瘟抗原发生特异性反应.本研究为非洲猪瘟亚单位疫苗研发及血清学检测方法的建立提供物质储备.
Abstract
To prepare monoclonal antibodies against the CP312R protein of African swine fever vi-rus(ASFV),BALB/c mice were immunized with eukaryotic expression of CP312R protein of Af-rican swine fever virus(ASFV),and the reactivity of the obtained monoclonal antibodies was iden-tified by indirect immunofluorescence assay(IFA).The vector pOET3-CP312R was constructed u-sing the baculovirus expression system(BES),and it was co-transfected with the baculovirus ge-nome flashBACTM ULTRA into Sf9 insect cells to obtain the recombinant baculovirus AcMNPV-CP312R.The virus expressed the recombinant ASFV CP312R protein in an intracellular soluble form.Identification by Western blot showed that the recombinant CP312R protein could react spe-cifically with ASFV positive serum.In addition,two monoclonal antibodies(McAb)targeting the recombinant CP312R protein were obtained.The titer of McAbs were detected by indirect ELISA,and was found to be no less than1∶1 024 000.IFA detection showed that monoclonal antibodies reacted specifically with ASFV.This study provides material reserves for the development of Afri-can swine fever subunit vaccines and the establishment of serological testing methods.
关键词
非洲猪瘟/CP312R蛋白/重组杆状病毒/单克隆抗体/间接ELISAKey words
African swine fever/protein CP312R/recombinant baculovirus/monoclonal antibody/in-direct ELISA引用本文复制引用
出版年
2024
NETL
NSTL
万方数据