稳定表达EGFR胞外区的NIH3T3细胞株的建立及鉴定  

Establishment and identification of NIH3T3 cells line stably expressing extracellular domain of EGFR

作　　者：江秀玲[1] 胡有根[2] 石必枝[3] 

机构地区：[1]扬州职业大学医学院,江苏扬州225000 [2]武警江苏总队医院泌尿外科,江苏扬州225003 [3]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：《现代医药卫生》2013年第3期327-328,330,共3页Journal of Modern Medicine & Health

摘　　要：目的构建稳定表达表皮生长因子受体(EGFR)胞外区(EGFRex)的NIH3T3细胞株。方法构建带有EGFRex基因的重组真核表达质粒PLNCX2-EGFRex,经脂质体法转染NIH3T3细胞后,用G418筛选阳性克隆,用免疫组化和蛋白质印迹(Western blot)检测EGFRex蛋白的表达,用EGF-EGFP蛋白检测其活性。结果成功构建了真核表达载体PLNCX2-EGFRex。以其转染NIH3T3细胞后,经免疫组化和Western blot检测到EGFRex的表达。在荧光显微镜下见细胞膜上有绿色荧光。结论人EGFRex在NIH3T3细胞内以其天然活性形式稳定表达,为抗体制备奠定了基础。Objective To establish NIH3T3 ceils line stably expressing extracellular domain of epidermal growth factor reeeptor（EGFRex）. Methods Recombinant eukaryotic expression plasmid PLNCX2-EGF Rex with EGFRex gene was constructed. After transfection to NIH3T3 cells by the lipofectin method,the positive clone was screened by G418. The expression level of EGFRex was examined by immunohistochemical staining and Western blot. The activity of EGFRex was examined by EGF-EGFP protein. Results The eukaryotic expression vector PLNCX2-EGFRex was successfully constructed. After transfetion to NIH3T3 cells, the expression of EGFRex was examined by Western blot and immunohischemieal staining. Green fluorescent was seen in the cell membrane by fluorescence microscope. Conclusion EGFRex is stably expressed as the natural active form in NIN3T3 cells, which can lay the foundation for antibody preparation.

关 键 词：表皮生长因子 NIH 3T3细胞 基因表达 表皮生长因子受体胞外区 

分 类 号：R730.2[医药卫生—肿瘤]