牛IFN-γ真核表达系统的构建及单克隆抗体的制备与鉴定  

Construction of Bovine IFN-γEukaryotic Expression System and Preparation and Identification of Monoclonal Antibodies

作　　者：许芳 蔡杰 薛华平 罗均[1] 蒋永青 郭霄峰[1] XU Fang;CAI Jie;XUE Hua-ping;LUO Jun;JIANG Yong-qing;GUO Xiao-feng(College of Veterinary Medicine,South China Agricultural University,Guangzhou,Guangdong,510642,China;Shenzhen Lvshiyuan Biotechnology Co.,Ltd,Shenzhen,Guangdong,518120,China)

机构地区：[1]华南农业大学兽医学院,广东广州510642 [2]深圳市绿诗源生物技术有限公司,广东深圳518120

出　　处：《动物医学进展》2024年第3期1-8,共8页Progress In Veterinary Medicine

基　　金：岭南现代农业科学与技术广东省实验室肇庆分中心“十四五”项目(P20211154-030303)。

摘　　要：为构建牛γ干扰素(IFN-γ)真核表达系统并制备单克隆抗体,将IFN-γ的基因克隆至pFastBacl载体上,构建pFastBacl-IFN-γ重组质粒,转化到DH10Bac后形成重组Bacmid,经过蓝白斑筛选将重组杆粒转染到Sf9细胞,经鉴定,获得了表达IFN-γ的重组杆状病毒;通过感染处于对数生长期的Sf9细胞,纯化细胞培养液上清获得了真核表达的牛IFN-γ蛋白。用纯化的IFN-γ蛋白免疫Balb/c小鼠,通过杂交瘤技术获得4株能稳定分泌的抗牛IFN-γ单克隆抗体的杂交瘤细胞株。经双抗夹心ELISA方法检测,结果显示4C3和4H11具有较好的夹心效果,为后续牛结核病IFN-γ体外释放ELISA检测试剂盒的研发奠定了基础。In order to construct the eukaryotic expression system of bovine interferon-gamma(IFN-γ)and develop the monoclonal antibodies,the gene of IFN-γwas cloned into pFastBacl vector to construct the recombinant plasmid pFastBacl-IFN-γ,which was transformed into DH10Bac to form recombinant Bacmid.The recombinant baculovirus expressing IFN-γwas obtained after blue-white plaque screening.The eukaryotic expressed bovine IFN-γprotein was obtained by infecting Sf9 cells in logarithmic phase and purifying the supernatant of cell culture medium.Balb/c mice were immunized with purified IFN-γprotein,and four hybridoma cell lines secreting stable anti-bovine IFN-γmonoclonal antibodies were obtained by hybridoma technique.The results of double antiboty sandwich ELISA showed that 4C3 and 4H11 had better sandwich effect,which laid a foundation for the development of ELISA detection kit for the IFN-γrelease in vitro of bovine tuberculosis.

关 键 词：牛γ干扰素 重组杆状病毒 夹心ELISA 

分 类 号：S852.4[农业科学—基础兽医学]