pICln蛋白单克隆抗体的制备及初步鉴定  

Preparation and identification of monoclonal antibody against pICln protein

作　　者：李晓梅[1] 李俐[1] 马颖哲[1] 郗艳丽[1] 张蒙[1] 

机构地区：[1]吉林大学白求恩医学院生物化学教研室、第二综合实验室,长春130021

出　　处：《中国免疫学杂志》2008年第7期640-643,共4页Chinese Journal of Immunology

基　　金：国家自然科学基金资助项目(30570688)

摘　　要：目的:制备特异性的抗pICln蛋白的单克隆抗体(McAb),为pICln的纯化及检测等提供必要的工具。方法:以pICln的合成肽为免疫原,对BALB/c小鼠注射免疫,将免疫鼠脾细胞和小鼠骨髓瘤细胞NS-1进行细胞融合,用显微镜下挑选单个克隆的克隆化方法克隆化,用间接ELISA检测法进行筛选。单抗的效价、亚型及特异性用ELISA、免疫双向扩散实验和Western blot等检测。结果:获得了稳定的分泌型杂交瘤细胞株大2B7和4A3,2B7培养上清液单抗效价为1∶64,属IgG1亚型,其腹水抗体效价及纯化抗体的效价分别为1∶1.28×104、1∶6.4×103,Western blot及免疫耗竭实验呈现一条约40kD的特异性带,免疫组织化学染色显示在细胞核和细胞质中呈现阳性反应。结论:本次制备的单抗具有特异性、高效价,可应用于pICln蛋白的纯化及检测等研究。Objective：To prepare the specific McAb against pICln for its utility. Methods： BALB/c mice were immunized using the synthetic peptide of pICln. Spleen cells of the mice were fused with NS- 1 mouse myeloma cells, and the bybridoma clone were selected under microscope, and screened by indirect ELISA. The titer, isoform and specificity of the McAb were tested through ELISA, two direction immune diffusion of agar and Western blot. Results： 2B7 and 4A3 hybridoma cell lines, which secreted McAbs against pICln, were successfully obtained. The titer of 2B7 culture supematants was 1 ： 64. The immunoglobulin of the McAb was classified as IgG1. The titer of ascetic fluid and the purified McAb were 1 ： 1.28 × 10^4 and 1 ： 6.4 ×10^3 respectively. A single band of pICln immune complex was showed about 40 kD position, and the antigen recognioed was present in cell nuclei and cytoplasm. Conclusion： The McAb has specificity and is produced with higher titer, which can be used for studying on pICln.

关 键 词：pICAn 合成肽 单克隆抗体 WESTERN BLOT 

分 类 号：R392.11[医药卫生—免疫学]