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本研究目的在于构建含内分泌及外分泌蛋白(EECP)基因及增强型绿色荧光蛋白(EGFP)基因非融合表达的真核表达载体,并转染乳腺癌细胞株MCF-7,检测EECP基因在真核细胞中的表达,初步研究EECP基因对细胞的生物学影响。EECP全长序列克隆至载体pBluescriptⅡSK(+)上,酶切连接至pIRES2-EGFP载体后转化大肠杆菌,筛选阳性克隆进行酶切电泳鉴定及测序鉴定。重组质粒转染MCF-7细胞,筛选细胞克隆MCF-7/MOCK及MCF-7/EECP,提取总蛋白确定EECP蛋白表达量变化。检测MCF-7/MOCK、MCF-7/EECP两组细胞增殖、细胞周期的变化。结果表明,重组质粒经酶切和测序证明构建正确,能有效转染乳腺癌细胞株MCF-7且EECP蛋白的表达量增加;MCF-7/EECP细胞较MCF-7/MOCK细胞增殖加快。结论:成功构建了乳腺癌相关新基因EECP与内部核糖体进入位点(IRES)连接的EGFP基因双元表达载体pEECP-IRES2-EGFP,为进一步研究EECP基因的生物学功能奠定了基础。
The purpose of this study was to construct an eukaryotic expression vector containing non-fusion expression of endocrine and exocrine protein (EECP) gene and enhanced green fluorescent protein (EGFP) gene and to transfect breast cancer cell line MCF-7 to detect the expression of EECP gene in eukaryotic Cells in order to study the biological effects of EECP gene on cells. EECP full-length sequence was cloned into the vector pBluescriptⅡSK (+), digested with pIRES2-EGFP vector and transformed into E.coli. The positive clones were screened for enzyme digestion electrophoresis and sequencing. The recombinant plasmids were transfected into MCF-7 cells, and the cell clones MCF-7 / MOCK and MCF-7 / EECP were screened. The total protein was extracted to determine the expression of EECP protein. The proliferation and cell cycle of MCF-7 / MOCK and MCF-7 / EECP groups were detected. The results showed that the recombinant plasmids were constructed correctly and confirmed by restriction enzyme digestion and sequencing. The recombinant plasmids could efficiently transfect breast cancer cell line MCF-7 and the expression of EECP protein increased. MCF-7 / EECP cells proliferated faster than MCF-7 / MOCK cells. Conclusion: The EGFP gene binary expression vector pEECP-IRES2-EGFP linked with the new ribosome entry site (IRES), a novel gene associated with breast cancer, was successfully constructed, which laid the foundation for further study on the biological function of the EECP gene.