稳定表达EGFRvⅢex的NIH3T3细胞株的建立及其免疫原性分析  

Establishment of a NIH3T3 cell line stably expressing EGFRvⅢ extracellular domain and evaluation of its immunogenicity

作　　者：江秀玲[1] 毕富勇[1] 王华茂[2] 石必枝[2] 张捷[2] 汪茗[1] 李宗海[2] 

机构地区：[1]皖南医学院生物化学教研室,安徽芜湖241001 [2]上海市肿瘤研究所癌基因及相关基因国家重点实验室,上海200032

出　　处：《细胞与分子免疫学杂志》2007年第7期612-615,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：上海市科委重点资助项目(054119568)

摘　　要：目的:建立表皮生长因子突变体Ⅲ胞外区(EGFRvⅡ-Iex)的NIH3T3稳定细胞系并分析其免疫原性。方法:将编码EGFRvⅢex基因的表达质粒pLNCX2-EGFRvⅢex转染NIH3T3细胞后,用G418筛选阳性克隆,得到多个细胞克隆。采用免疫组化和Western blot法鉴定这些细胞克隆。选取高表达EG-FRvⅢex的细胞株(命名为3T3-vⅢex)免疫BALB/c小鼠,制备抗血清。用ELISA、Western blot和免疫荧光分别检测抗血清的效价和特异性。结果:成功地构建了真核表达载体pLNCX2-EGFRvⅢex并获得了稳定高表达EGFRvⅢex的NIH3T3细胞系3T3-vⅢex。用3T3-vⅢex免疫小鼠所获得的抗血清效价为10-5。Western blot和免疫荧光鉴定证明抗血清可以与EGFR-vⅢex特异结合。结论:人EGFRvⅢex可在NIH3T3细胞内获得稳定表达,以其免疫小鼠可以获得高效价、高特异性的抗血清。AIM： To establish a cell line stably expressing EGFRvlIlex （epidermal growth factor receptor variant Ⅲ extracellular domain） and evaluate its immunogenicity. METHODS： NIH3T3 cells stably expressing EGFRvⅢex was obtained by screening NIH3T3 cells transfected with pLNCX2-EGFRvⅢex, a plasmid encoding EGFRvlIlex. The expression level of EGFRvⅢex was examined by immuno- histochemical staining and Western blot. The cell clone with the highest expression of EGFRvlIlex named as 3T3-vlllex was used to immunize BALB/c mice. The titer and specificity of murine antiserum was evaluated by ELISA, Western blot and immunofluorescent staining. RESULTS： A NIN3T3 stable cell line with high EGFRvlIlex expression was obtained. The titer of the antiserum against human EGFRvlIlex was about 10-5 Western blot and immunofluorescent staining analysis revealed the high specificity of the antiserum. CONCLUSION： High titer of antiserum against EGFRvⅢex can be obtained by NIH3T3 cell lines stably expressing EGFRvⅢex.

关 键 词：NIH3T3 表皮生长因子受体突变体Ⅲ 稳定细胞系 免疫原性 抗体 

分 类 号：R392.11[医药卫生—免疫学]