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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 ,并为制备抗EpCAM的mAb提供了免疫原
OBJECTIVE: To construct eukaryotic expression vector of pIg EpCAM and pEGFP EpCAM and express in COS7 cells. Methods: The EpCAM cDNA was amplified by PCR, ligated with pIg and pEGFP, respectively, and transfected into COS7 cells by lipofectamine. The expression products of pIg EpCAM were detected by immunoprecipitation. The expression of EpCAM GFP was observed by fluorescence microscopy. Results: The results of DNA sequencing showed that pIg EpCAM and pEGFP EpCAM vectors were constructed correctly. The result of immunoprecipitation showed that the culture supernatant of COS7 cells transfected with pIg EpCAM contained a fusion protein with a relative molecular mass (Mr) of 650,000 which showed good immunoreactivity with anti-human IgGFc mAb . Fluorescence microscopy showed that the green fluorescence of COS7 cells transfected with empty vector pEGFP was uniformly distributed in the cytoplasm and nucleus. The green fluorescence of COS7 cells transfected with pEGFP EpCAM mainly distributed on the cell surface. CONCLUSION: Two eukaryotic expression vectors for EpCAM were successfully constructed and expressed in COS7 cells, which provided the conditions for the functional study of EpCAM and provided the immunogen for the preparation of anti-EpCAM mAb