非洲猪瘟病毒F334L基因编码蛋白的原核表达及多克隆抗体制备  

Prokaryotic Expression of African Swine Fever F334L Gene Expressing Protein and Preparation of Its Polyclonal Antibody

作　　者：刘佳晨 李丽薇[1,2] 郭子强 陈金霞 曹云雷 乔思娜 刘长龙 赵冉[3] 童光志 高飞[1,2] LIU Jiachen;LI Liwei;GUO Ziqiang;CHEN Jinxia;CAO Yunlei;QIAO Sina;LIU Changlong;ZHAO Ran;TONG Guangzhi;GAO Fei(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonosis,Yangzhou University,Yangzhou 225009,China;Xiamen Center for Animal Disease Control and Prevention,Xiamen 361009,China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]扬州大学江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州225009 [3]厦门市动物疫病预防控制中心,厦门361009

出　　处：《中国动物传染病学报》2024年第4期173-178,共6页Chinese Journal of Animal Infectious Diseases

基　　金：国家重点研发计划课题(2021YFD1801401);国家自然科学基金ASFV专项(31941017);中央级公益性科研院所基本科研业务费专项(Y2020YJ15);上海市自然科学基金项目(21ZR1476900,21ZR1477200)。

摘　　要：本研究通过将非洲猪瘟病毒(African swine fever virus,ASFV)的一种非结构蛋白编码基因F334L克隆至原核表达载体pCold-Ⅰ,完成重组表达质粒pCold-Ⅰ-ASFV-F334L的构建。将pCold-Ⅰ-ASFV-F334L转化受体菌感受态细胞,经1 mmol/L的IPTG诱导原核表达。SDS-PAGE和Western blot试验检测结果表明,F334L基因得到了良好的表达,将所得的F334L基因编码蛋白纯化后免疫小鼠,获得含多克隆抗体的血清。利用该抗体检测实验室构建并拯救的表达ASFV F334L基因编码蛋白的重组猪繁殖与呼吸综合征病毒rPRRSV-F334L,试验结果显示该多克隆抗体具有良好的反应原性和特异性,证明了ASFV F334L基因可在PRRSV活载体中稳定表达,为ASFV活载体疫苗的研发提供了材料。The recombinant expression plasmid pCold-Ⅰ-ASFV-F334L was constructed by cloning the F334L gene encoding one of non-structural protein of African swine fever virus(ASFV)into the prokaryotic expression vector pCold-Ⅰ.The pCold-Ⅰ-ASFV-F334L was transformed into competent cells,and the prokaryotic expression was induced by 1 mmol/L IPTG.Through SDS-PAGE and Western blot experiments,the results indicated that the F334L gene was well expressed.The purified F334L gene-encoded protein was used to immunize mice,resulting in the production of polyclonal antibodies in the serum.The generated antibodies were used to detect the recombinant porcine reproductive and respiratory syndrome virus expressing ASFV F334L gene-encoded protein(rPRRSV-F334L)constructed and rescued by the research team.The experimental results demonstrated that the polyclonal antibodies exhibited good reactivity and specificity,proving that the ASFV F334L gene can be stably expressed in the PRRSV live vector,thereby providing material for the development of ASFV live vector vaccines.

关 键 词：非洲猪瘟病毒 病毒活载体 F334L基因 重组病毒 

分 类 号：S852.651[农业科学—基础兽医学]