非洲猪瘟病毒p22蛋白单克隆抗体制备及其抗原表位鉴定  

Preparation of Monoclonal Antibody Against African Swine Fever Virus p22 Protein and Identification of the Antigenic Epitope

作　　者：王小格 司煊瑛 闫志伟[1,2] 王飞 尤龙琪 刘梗 蔡茂 梁珺珹 梁玉秀 杜永坤 张改平 WANG Xiaoge;SI Xuanying;YAN Zhiwei;WANG Fei;YOU Longqi;LIU Geng;CAI Mao;LIANG Juncheng;LIANG Yuxiu;DU Yongkun;ZHANG Gaiping(International Joint Research Center of National Animal Immunology,College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China;Henan Provincial Animal Biological Products Engineering Research Center,Zhengzhou 450046,China;Animal Disease Prevention and Control Center,Agriculture and Rural Bureau,Fugu County,Shaanxi Province,Yulin 719499,China)

机构地区：[1]河南农业大学动物医学院,国家动物免疫学国际联合研究中心,郑州450046 [2]河南省动物生物制品工程研究中心,郑州450046 [3]陕西省府谷县农业农村局动物疫病预防控制中心,榆林719499

出　　处：《中国畜牧兽医》2025年第2期781-791,共11页China Animal Husbandry & Veterinary Medicine

基　　金：河南省重大科技专项(221100110600);河南省自然科学基金面上项目(222300420460);中国博士后科学基金面上项目(2021M690926)。

摘　　要：[目的]采用原核表达系统表达并获得非洲猪瘟病毒(ASFV)p22重组蛋白,并进一步制备、鉴定p22重组蛋白的单克隆抗体。[方法]采用PCR方法扩增ASFV p22蛋白编码基因,构建重组表达质粒pET-28a(+)-p22,并通过IPTG诱导表达p22蛋白。将纯化后重组蛋白免疫小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行融合制备单克隆抗体。通过Western blotting鉴定单克隆抗体特异性,通过ELISA方法测定腹水效价;利用在线软件预测p22蛋白抗原表位分布情况并合成重叠多肽,通过Dot blotting鉴定抗体识别的抗原表位。[结果]本研究成功构建了p22重组蛋白的原核表达质粒,获得原核系统表达的p22重组蛋白,分子质量约为22 ku。用纯化后p22蛋白作为免疫原免疫小鼠,成功筛选到4株阳性单克隆杂交瘤细胞株,分别为1G3D7G11、2G5H6H8、6D10G7D6和8F6F8B9,其腹水效价均达到1∶500 000。Western blotting结果显示,4株单克隆抗体均能与p22蛋白发生特异性反应。单克隆亚型鉴定结果显示,1G3D7G11、2G5H6H8和6D10G7D6株单克隆抗体的重链均为IgG1类,8F6F8B9株单克隆抗体的重链为IgG2a类,4株单克隆抗体轻链均为Kappa型。Dot blotting检测结果显示,1G3D7G11、2G5H6H8株单克隆抗体识别抗原表位位于第58—89位氨基酸处;8F6F8B9株单克隆抗体识别抗原表位位于第126—150位氨基酸处。[结论]本研究成功制备了4株ASFV p22蛋白的单克隆抗体,并初步鉴定了单克隆抗体所识别的B细胞表位区间。研究结果为p22蛋白的生物学功能研究和ASFV血清学检测提供了参考依据。[Objective]The purpose of this study was to express and obtain recombinant protein p22 of African swine fever virus(ASFV)using the prokaryotic expression system,and further prepare and identify the monoclonal antibody against recombinant protein p22.[Method]PCR was used to amplify the ASFV p22 protein coding gene and construct the recombinant expression plasmid pET-28a(+)-p22,and induced to express p22 protein by IPTG.The purified recombinant protein was immunized to mice,and the spleen cells of immunized mice were fused with SP2/0 myeloma cells to prepare monoclonal antibody.The specificity of monoclonal antibody was determined by Western blotting,and the titer of ascites was determined by ELISA.Online software was used to predict the distribution of p22 protein epitopes and synthesize overlapping peptides,and the epitopes recognized by antibodies were identified by Dot blotting.[Result]In this study,the prokaryotic expression plasmid of p22 recombinant protein was successfully constructed,and the recombinant protein p22 expressed in the prokaryotic system was obtained with a molecular mass of about 22 ku.After immunizing mice with purified p22 protein as immunogen,4 monoclonal hybridoma cell lines were successfully screened,which was 1G3D7G11,2G5H6H8,6D10G7D6 and 8F6F8B9,and the ascites titer reached 1∶500000.Western blotting results showed that 4 monoclonal antibodies could react specifically with p22 protein.The identification results of monoclonal subtypes showed that the heavy chains of monoclonal antibodies of 1G3D7G11,2G5H6H8 and 6D10G7D6 were IgG1,the heavy chain of monoclonal antibody of 8F6F8B9 was IgG2a,and the light chains of the 4 monoclonal antibodies were Kappa.Dot blotting test results showed that the epitopes of the recognition antigen of monoclonal antibodies 1G3D7G11 and 2G5H6H8 were located at amino acid 58-89.The epitope of monoclonal antibody 8F6F8B9 was located at amino acid 126-150.[Conclusion]In this study,4 monoclonal antibodies against ASFV p22 protein were successfully prepared,and

关 键 词：非洲猪瘟病毒 p22蛋白 单克隆抗体 抗原表位 

分 类 号：S852.651[农业科学—基础兽医学]