基因疫苗脾内免疫制备抗人c-kit单克隆抗体及其初步鉴定  

Production of anti-human c-kit monoclonal antibodies by direct intra-spleen injection of DNA vaccine

作　　者：石琳[1] 何大水[1] 冯春玲[1] 袁向飞[1] 屈浩[1] 黄丽华[1] 张丽艳[1] 王冬梅[1] 张毅[1] 张宇光[1] 

机构地区：[1]中国医学科学院中国协和医科大学血液学研究所血液病医院,天津300020

出　　处：《中国免疫学杂志》2008年第7期644-648,651,共6页Chinese Journal of Immunology

摘　　要：目的:用基因脾内免疫方法制备抗人c-kit单克隆抗体,并通过对抗体生物学特性的初步研究,确定该方法制备抗体的可行性。方法:利用分子克隆方法构建重组质粒pcDNA3.1/c-kit胞外区,脾内免疫BALB/c小鼠一次,通过杂交瘤技术制备抗人c-kit单克隆抗体。采用流式细胞术、Western blot方法初步鉴定制备的抗体。结果:目的基因片段c-kit胞外区正确插入质粒pcDNA3.1,重组质粒免疫小鼠后通过杂交瘤技术获得了三株分泌抗人c-kit胞外区的杂交瘤细胞株6C4、2C5和5D5,其亚类均为IgM类,识别不同的抗原表位,其特异性与抗人c-kit抗体一致。结论:可用基因脾内一次免疫法制备抗人c-kit单克隆抗体。Objective： To prepare anti-human c-kit monoclonal antibody（McAb） by genetic immunization in spleen, and to determine practicability of these means to produce McAbs based on the biological activity of anti-human c-kit antibody. Methods： Recombinant plasmid PeDNA3.1/c-kit extracellular domain was constructed by molecular cloning techniques, and was used to immunize BALB/c mice in spleen directly to prepare mAb against human c-kit by routine hybridoma technique. FASC,fluorescence microscope and Western blot were utilized to i- dentify the prepared antibody. Results：c-kit extracellular region was cloned and insert peDNA3.1 plasmid successfully. Three hybridoma cell fines 6CA,2C5 and 5D5 that secrete anti-htunan c-kit McAbs were ohtained after using intra-spleen immunization with a DNA vaccine. The isotypes of these three antibodies were all IgM,and the epitopes were different with each other. Conclusion： The method of genetic immunization into spleen can be used to prepare anti-human c-kit monoclonal antibodies.

关 键 词：C-KIT 基因免疫 单克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]