摘要
目的 :构建pIg EpCAM及pEGFP EpCAM真核表达载体 ,并在COS7细胞中表达。方法 :用PCR扩增EpCAMcDNA ,酶切后分别与pIg及pEGFP两种载体连接 ,用脂质体法转染COS7细胞。用免疫沉淀法检测pIg EpCAM的表达产物 ;用荧光显微镜观察EpCAM GFP的表达。结果 :DNA序列测定的结果显示 ,pIg EpCAM及pEGFP EpCAM载体的构建正确。免疫沉淀的结果显示 ,转染pIg EpCAM的COS7细胞的培养上清中含有相对分子质量 (Mr)为 6 5 0 0 0的融合蛋白 ,该蛋白与抗人IgGFc段的mAb具有良好的免疫反应性。荧光显微镜观察可见 ,转染空载体pEGFP的COS7细胞中绿色荧光均匀地分布于胞质和胞核 ;而转染pEGFP EpCAM的COS7细胞中绿色荧光主要分布于细胞表面。结论 :成功地构建了两种EpCAM的真核表达载体 ,并在COS7细胞中表达 ,为EpCAM的功能研究创造了条件 。
AIM: To construct EpCAM eukaryotic expression vectors pIg-EpCAM and pEGFP-EpCAM and to express them in COS7 cells. METHODS: EpCAM cDNA was amplified by PCR and then inserted into the pIg and pEGFP vectors respectively to construct recombinant vectors pIg-EpCAM and pEGFP-EpCAM. The two recombinant vectors were transfected into COS7 cells under the mediation of liposome. The expressed EpCAM-Ig fusion protein was detected by Western blot. The expression of EpCAM-GFP fusion protein was observed under fluorescence microscope. RESULTS: DNA sequencing demonstrated that EpCAM was correctly cloned into the two vectors. The culture supernatant of the pIg-EpCAM-transfected COS7 cells could bind effectively to mAb against human IgG Fc. Green fluorescence distributed evenly in the cytoplasm and nuclei of pEGFP-transfected COS7 cells, whereas in pEGFP-EpCAM transfected COS7 cells, green fluorescence distributed mainly on the surface of the cells. CONCLUSION: Two EpCAM eukaryotic expression vectors have been constructed and expressed successfully, which lays the foundation for functional research of EpCAM and for preparation of mAb to EpCAM.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2004年第6期765-768,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家重点基础研究发展规划(973)资助项目 (No .2 0 0 1CB51 0 0 0 4 )
关键词
上皮细胞黏附分子
真核表达
增强型绿色荧光蛋白
epithelial cell adhesion molecule
eukaryotic expression
enhanced green fluorescence protein
