乳腺癌相关新基因EE-CP与IRES连接的EGFP基因双元表达载体的构建及初步功能研究  

Construction and Expression of Bisbicistronic Expression Vector of Novel Endocrine and Exocrine Protein Gene Associated with Breast Cancer and IRES Mediated Gene EGFP

作　　者：严华梅[1] 王艳萍[1] 王宇[1] 王竹[1] 郑鸿[1] 

机构地区：[1]四川大学华西医院肿瘤分子诊断研究室,生物治疗国家重点研究室,成都610041

出　　处：《生物医学工程学杂志》2012年第4期737-744,共8页Journal of Biomedical Engineering

基　　金：国家博士点基金资助项目(20090181110070)

摘　　要：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。This experimental study was aimed to construct the recombinant bisbieistronic eukaryotic expression vector eontaining endocrine and exocrine protein（EECP） gene associated with breast cancer and enhanced green fluorescent protein （EGFP） gene. And then we transfected it into breast cancer cells MCF-7 to detect the expression of EECP protein and study preliminary biological function of EECP gene. The EECP sequence was cloned to pBlueseript II SK （4-） plasmid. After restriction endonuclease reaction of pBluescript ]1 SK（4-） plasmid, the EECP fragment was cloned to plRES2-EGFP vector forming a recombinant eukaryotic expression vector named pEECP-IRES2-EGFP. The potential vector was identified by restriction endonuclease digestion and sequencing. Correct plasmid was extrae- ted and transfected into breast cancer cells MCF-7. The expression of EECP protein was detected by western blot a- nalysis. Its biological function was studied by MTT and Flow-cytometry. It turns out that the recombinant eukaryot- ic expression vector containing EECP gene and EGFP gene was constructed successfully, and it could transfect MCF- 7 cells efficiently. It can get higher expression of EECP protein and higher cell proliferation, thus providing an im- portant and convenient tool for studying the function of EECP gene in vitro and in vivo.

关 键 词：内分泌及外分泌蛋白 内部核糖体进入位点 增强型绿色荧光蛋白 乳腺癌 

分 类 号：R737.9[医药卫生—肿瘤]