一株分泌抗猪γ-干扰素单克隆抗体的杂交瘤细胞的鉴定  

Identification of a Strain of Hybridoma Cell Secreting the Monoclonal Antibody against Porcine IFN-γ

作　　者：张敬梅[1] 余为一[1] 李林[1] 张春玲[1] 

机构地区：[1]安徽农业大学安徽省人兽共患病重点实验室,安徽合肥230036

出　　处：《安徽农业科学》2009年第27期12955-12956,12983,共3页Journal of Anhui Agricultural Sciences

基　　金：安徽省科技攻关项目(04013042)资助

摘　　要：[目的]应用杂交瘤技术获得稳定分泌抗猪γ-干扰素单克隆抗体的杂交瘤细胞株。[方法]先将原核表达产物His-PoIFN-γ免疫BALB/c小鼠4次,然后进行细胞融合,再以纯化的GST-PoIFN-γ作为包被抗原,用间接ELISA筛选阳性杂交瘤无性系,最终获得能稳定分泌抗猪IFN-γ单克隆抗体的杂交瘤细胞株,对其进行间接ELISA和Western blot检测。[结果]试验获得1株能稳定分泌抗猪IFN-γ单克隆抗体的杂交瘤细胞株,被命名为B3株,经间接ELISA和Western blot检测,该株分泌的抗体能与融合蛋白His-PoIFN-γ和GST-PoIFN-γ发生特异性反应,而与其他表达鸡γ-干扰素、His和GST蛋白等的重组质粒均不发生反应。B3株分泌的抗体小鼠腹水的ELISA效价达到1∶160 000,抗体亚型为IgG1型,且轻链为κ链。[结论]该研究为建立猪γ-干扰素检测方法,研究机体的免疫状态及免疫机制提供了技术基础。[ Objective ] The study aimed to obtain the strain of hybridoma cell that was stable to secrete the monoclonal antibody （mAb） a- gainst porcine IFN-γ. [ Method] BALB/e mice were first immunized for 4 times with prokaryotic expression protein His-PolFN--γand then SP2/ 0 myeloma cells were fused with spleen cells of the immunized mice according to standard procedure. And then, the purified GST-PolFN-5, was used as the detecting antigen and the positive hybridoma clones were screened by indirect ELISA and limited dilutions. Finally, a hybridoma which stably secreted the specific mAb against porcine IFN-γwas obtained and its specificity was characterized by indirect ELISA and Western blot. [ Result] A hybridoma which stably secreted the specific mAb against porcine IFN-γ was obtained in this experiment and named as B3 strain. The detection by indirect ELISA and Westem blot showed that the mAb secreted by B3 strain could only react with the fused proteins of His-PolFN-γand GST-PolFN-5,and it could not react with ChlFN-γ, aHis and GST expressed in E. coli. The ELISA titers of the mAb mice ascites secreted by B3 strain were 1：160 000, mAb subtype belonged to IgG1 type and the light chain was K chain. [ Conclusion] This study provided the technique foundation for establishing the detection method of porcine IFN-γand studying the immunization statue and mechanism of the organism.

关 键 词：猪Γ-干扰素 融合蛋白 单克隆抗体 制备 鉴定 

分 类 号：S852[农业科学—基础兽医学]