ECM1-pEGFP—N2真核表达载体的构建及其在人乳腺癌细胞系MCF-7中的表达  

Construction of extracellular matrix protein 1 in eukaryotic expression vector and its expression in breast cancer cell line MCF-7

作　　者：侯彦强[1] 娄加陶[2] 彭亮[1] 倪健 孔宪涛[2] 仲人前[2] 

机构地区：[1]上海市松江区中心医院检验科,上海201600 [2]第二军医大学长征医院实验诊断科、全军临床免疫中心,上海200003 [3]上海富纯中南生物技术有限公司,上海201702

出　　处：《世界肿瘤杂志》2006年第4期233-236,252,共5页Tumour Journal of the World

基　　金：国家自然科学基金项目（30080027）、上海市松江区科学技术攻关项目（2006-27）

摘　　要：目的构建细胞外基质蛋白1基因（ECM1）的真核表达载体ECM1-pEGFP-N2，检测其在人乳腺癌细胞系MCF-7中表达。方法采用PCR方法，以ECMleDNA为模板，扩增出ECM1基因，用Bg1 Ⅱ和KpnⅠ双酶切ECM1基因和pEGFP-N2载体，连接酶切目的片段，转化大肠杆菌DH5，筛选阳性克隆酶切、测序鉴定；利用脂质体介导的转染技术转染MCF-7细胞，荧光显微镜检测报告基因表达产物EGFP，免疫组化检测ECM1蛋白表达。结果利用PCR克隆出约1．6kb的ECM1基因；PCR鉴定、酶切鉴定及测序结果证实成功构建ECM1-pEGFP-N2真核表达载体；转染MCF-7细胞后，可在细胞内观察到报告基因产生的绿色荧光，用免疫组化方法可检测到ECM1蛋白。结论成功构建了ECM1-pEGFP-N2真核表达载体，并在MCF-7细胞中表达，为进一步研究ECM1的功能奠定了基础。Objective To construct a human extracellular matrix protein 1 （ECM 1） in eukaryotic expression vector ECMI-pEGFP-N2 and detect whether it could be expressed in MCF-7 cells. Methods The methods of PCR and double enzymes （Bgl Ⅱ and Kpn Ⅰ ） cutting were used, The complete ECM1 gene was obtained from ECM1 cDNA. The recombinant of gene was transformed into E.coli.DH5, the positive cloned were selected and the plasmid DNA was identified by restriction enzyme analysis and sequencing; ECMI-pEGFP- N2 was transfected with Lipofectamine to MCF-7 cells, and the expression of ECMI-pEGFP-N2 was determined by GFP fluorescence and anti-ECM1 immunohistocbemistry. Results About 1.6kb ECM1 gene was successfully cloned. Identified by PCR, double enzymes digest and sequencing, these results showed that ECM1 eukaryotic expression vector ECMI-pEGFP-N2 was exactly constructed; Green fluorescence of fuse-protein-expression was found in MCF-7 cells, ECM1 protein could be detected in MCF-7 cells by immunohistochemical staining method. Conclusions We successfully construct ECM1 eukaryotic expression vector and it can be expressed in MCF-7 cells, which forms an important basis for the further studies on the roles of ECM 1.

关 键 词：细胞外基质蛋白1 绿色荧光蛋白 真核表达载体 

分 类 号：R737.9[医药卫生—肿瘤] R-33[医药卫生—临床医学]