鸡IFN-γ单克隆抗体的制备及其识别区的鉴定  

Development of monoclonal antibodies against chicken IFN-γ(chIFN-γ) and identification of their recognizing regions

作　　者：柳亚楠 王永强[1] 李晓齐[1] 曹红[1] 郑世军[1] 

机构地区：[1]中国农业大学动物医学院农业生物技术国家重点实验室,北京100193

出　　处：《中国兽医科学》2017年第6期721-726,共6页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31272543;31430085);现代农业产业技术体系建设专项(NYCYTX-41)

摘　　要：IFN-γ是鸡体内一种重要的细胞因子,为制备鸡IFN-γ的单克隆抗体,以ch IFN-γ-His融合蛋白免疫BALB/c小鼠,经3次免疫后分离出脾细胞与骨髓瘤细胞系(SP2/0)进行融合,经单克隆培养,筛选得到4株能稳定分泌单抗的杂交瘤细胞株,分别命名为2A10、2B10、4D10和4F9。应用间接ELISA方法鉴定4株单抗的重链类型分别为IgG2a、IgG1、IgG1和IgG2b。通过间接ELISA方法测定4株单抗的亲和力解离常数(Kd)分别为1.06×10^(-10)、7.35×10`(-10)、1.10×10^(-10)和9.34×10^(-10),表明4株单抗均为高亲和力抗体。将ch IFN-γ的C端、N端同时进行表达,应用Western-blot方法鉴定4株单抗的抗原识别表位分别为chIFN-γ的47-100位氨基酸,101-145位氨基酸和1-46位氨基酸。应用Western-blot方法鉴定4株单抗的特异性,4株单抗均能识别原核表达的重组蛋白chIFN-γ,而不识别鸡ch IL-2、ch IL-4、ch IL-10蛋白,表明4株单抗特异性良好。原核表达chIFN-γ蛋白制备单克隆抗体效果良好,为进一步研究chIFN-γ的功能奠定了基础。Chicken IFN-γ（chIFN-γ） plays an important role in host, To develop monoclonal antibodies （McAbs）against chIFN-γ,chIFN-γ-His recombinant protein was used to vaccinate BALB/c mice three,of which the spleen cells were fused with the separated splenocytes with myeloma （SP2/0）cells,cloned, cultured and screened. 4 hybridoma cell lines that stably secreted McAbs against chIFN-γ were screened and named as 2A10,2B10,4D10 and 4F9,respectively. The dissociation constant（Kd）of these McAbs were det- ermined and their Kdswere 1.06 ×10^-10,7,35×10^-10, 10 ×10^-10 and 9.34 ×10^-10,respectively. The isotypes of these McAbs were IgG2a,IgG1,IgG1 and IgG2b,respectively. Western-blot analysis demonstrated that chIFN-ywas recognized by 2A10,2B10,4D10,4F9 and targeted between amino acids 47 to 100,101 to 145 and i to 46,respectively,but not by chIL-2,chIL-4 and chIL-10. These McAbs would help to analyze the biological activities of chIFN-γ

关 键 词：鸡IFN-γ 原核表达 单克隆抗体 

分 类 号：S852.42[农业科学—基础兽医学]