非洲猪瘟病毒p30-54融合蛋白基因的构建、表达及其多克隆抗体制备  

Construction,Expression of p30-54 Fusion Protein Gene of African Swine Fever Virus and Preparation of Polyclonal Antibody

作　　者：李杰[1] 张星星[2] 郭晶 孟庆玲[1] 乔军[1] 张国武 王晓婷 李妍 才学鹏[3] LI Jie;ZHANG Xingxing;GUO Jing;MENG Qingling;QIAO Jun;ZHANG Guowu;WANG Xiaoting;LI Yan;CAI Xuepeng(Department of Animal Science&Technology,Shihezi University,Shihezi 832003,China;State Key Laboratory for Sheep Genetic Improvement and Healthy Production,Xinjing Academy of Agricultural and Reclamation Science,Shihezi 832000,China;Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832003 [2]新疆农垦科学院省部共建绵羊遗传改良与健康养殖国家重点实验室,新疆石河子832000 [3]中国农业科学院兰州兽医研究所,甘肃兰州730046

出　　处：《河南农业科学》2018年第9期126-130,共5页Journal of Henan Agricultural Sciences

基　　金：国家重点研发计划项目(2017YFD0502300);兵团中青年科技创新领军人才计划项目(2016BC001)

摘　　要：为了制备反应原性良好的非洲猪瘟病毒(ASFV)血清学诊断抗原,根据ASFV p30和p54基因序列,通过大肠杆菌密码子优化后分别体外合成全长的p30和p54基因,利用重叠延伸PCR(SOE-PCR)将2个基因片段串联,构建p30-54融合基因。将构建的融合基因片段克隆入p ET-28a(+)表达载体中,构建原核表达载体p ET-p30-54,转化至E. coli BL21(DE3)感受态细胞中,用IPTG诱导表达,检测融合蛋白的反应原性,并制备抗该融合蛋白的多克隆抗体。SDS-PAGE检测结果显示,表达的p30-54融合蛋白分子质量为47. 1 ku; Western blot分析显示,该融合蛋白可被ASFV阳性血清特异性识别,表明其具有良好的反应原性。将p30-54融合蛋白用Ni-NTA亲和层析柱纯化后免疫小鼠,成功制备了抗ASFV p30-54融合蛋白多克隆抗体。In order to prepare the serological diagnostic antigen of ASFV,p 30 and p 54 gene of ASFV were optimized and synthesized in vitro by using the E.coli codons according to ASFV p 30 and p 54 gene sequences.p 30-54 gene was constructed through SOE-PCR.Then fusion gene was cloned into a pET-28a(+)expression vector to generate expression vector pET-p 30-54.The recombinant pET-p 30-54 was transformed into E.coli BL21(DE3)for expression under the induction of IPTG.The reactiongenicity of recombinant protein was confirmed by Western blot,and this recombinant protein’s polyclonal antibody was prepared.The results of SDS-PAGE showed that recombinant protein p30-54 was successfully expressed in E.coli with a molecular mass of 47.1 ku.Western blot analysis showed that the recombinant protein could be specifically identified by ASFV-positive serum,which proved that it had good reactiongenicity.Polyclonal antibody against ASFV p30-54 fusion protein were prepared in immunized mice after the protein was purified with Ni-NTA affinity column.

关 键 词：非洲猪瘟病毒 p30-54融合蛋白 大肠杆菌表达系统 多克隆抗体 反应原性 

分 类 号：S852.659.1[农业科学—基础兽医学]