非洲猪瘟病毒p22蛋白单克隆抗体的制备及鉴定  

Preparation and Identification of Monoclonal Antibody against p22 Protein of African Swine Fever Virus

作　　者：矫健 李建达[2] 韩先杰[1] 张琳[2] 张玉玉[2] 任素芳[2] 刘飞 陈智[2] Nataliia Hrabchenko 张文娟 于江[1,2] 吴家强[1,2] Jiao Jian;Li Jianda;Han Xianjie;Zhang Lin;Zhang Yuyu;Ren Sufang;Liu Fei;Chen Zhi;Nataliia Hrabchenko;Zhang Wenjuan;Yu Jiang;Wu Jiaqiang(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao 266109,China;Institute of Animal Science and Veterinary Medicine,Shandong Academy of Agricultural Sciences/Shandong Key Laboratory of Animal Disease Control and Breeding/Key Laboratory of Livestock and Poultry Multi-omics,Ministry of Agriculture and Rural Affairs,Jinan 250100,China;Shandong Green Blue Biotechnology Company Limited,Taian 271400,China)

机构地区：[1]青岛农业大学动物医学院,山东青岛266109 [2]山东省农业科学院畜牧兽医研究所/山东省畜禽疫病防治与繁育重点实验室/农业农村部畜禽生物组学重点实验室,山东济南250100 [3]山东碧蓝生物科技有限公司,山东泰安271400

出　　处：《山东农业科学》2023年第10期140-145,共6页Shandong Agricultural Sciences

基　　金：山东省重点研发计划项目(2020CXGC010801,2022TZXD0041,2022CXPT010,2021LZGC001);山东省现代农业产业技术体系(SDAIT-08-01);山东省农业科学院农业科技创新工程项目(CXGC2023F10,CXGC2023A21,CXGC2023A21);泰山学者工程项目。

摘　　要：非洲猪瘟(African swine fever, ASF)是由非洲猪瘟病毒(African swine fever virus, ASFV)引起的一种高度传染性病毒性疾病,发病率和死亡率高达100%,对我国的生猪养殖业造成了毁灭性打击。p22蛋白为ASFV的结构蛋白,本研究构建了重组原核表达质粒pET-32a-p22,将其转化至大肠杆菌BL21(DE3)感受态细胞表达重组p22蛋白并纯化,经Western blot鉴定证实重组p22蛋白与ASFV阳性血清有良好的免疫反应性。利用重组p22蛋白免疫Balb/c小鼠并通过间接ELISA方法筛选到2株能够稳定分泌p22蛋白单克隆抗体(MAb)的杂交瘤细胞株2D6和5E7。经抗体亚型鉴定两株抗体均为IgG1型;经Western blot鉴定,两株抗体能够特异性识别过表达的p22蛋白。综上,本研究成功表达并纯化了p22重组蛋白,制备了p22单克隆抗体,为进一步探讨p22蛋白的结构功能及其在ASFV中的感染致病机制提供了基础条件。African swine fever(ASF)is a highly contagious viral disease caused by African swine fever virus(ASFV)with the morbidity and mortality up to 100%,which has caused a devastating blow to China’s pig farming industry.The p22 protein is a structural protein of ASFV.In this study,recombinant prokaryotic expression plasmid pET-32a-p22 was constructed and transformed into Escherichia coli BL21(DE3)receptive cells to express the recombinant p22 protein and purify it.Western blot analysis confirmed that the recombinant p22 protein had good immunore activity with ASFV positive serum.Balb/c mice were immunized with the re-combinant p22 protein and two hybridoma cell lines 2D6 and 5E7,which could secrete monoclonal antibody(MAb)to p22 protein,were screened by indirect ELISA.The antibody subtypes of the two strains were identi-fied as IgG1 type.Western blot analysis showed that the two antibodies could specifically recognize the overex-pressed p22 protein.In conclusion,the recombinant p22 protein was successfully expressed and purified in this study,and the p22 monoclonal antibody was prepared,which provided one of the basic conditions for fur-ther investigation of the structure and function of p22 protein,and also its role in the pathogenesis of ASFV in-fection.

关 键 词：非洲猪瘟病毒 p22蛋白 单克隆抗体 原核表达 

分 类 号：S852.651[农业科学—基础兽医学]