CD31启动子表达载体的构建与功能鉴定

Construction and Identification of Expression Vectors with CD31 Promotor

目的:构建CD31启动子的绿色荧光蛋白和萤光素酶真核表达载体,转染至内皮细胞系HUVEC和BEND3细胞中,对其生物学功能和活性进行初步检测,作为鉴定内皮细胞的新方法。方法:从基因组中钓取CD31启动子的核酸序列,将其插入pGL4.0萤光素酶载体和慢病毒表达载体pCDH-copGFP,经限制性内切酶酶切和测序验证后瞬时转染HUVEC和BEND3内皮细胞系及对照MCF7乳腺癌细胞,通过双萤光素酶实验检测其启动子活性,荧光显微镜观察绿色荧光表达。结果:双酶切与测序结果表明表达载体中正确插入了CD31启动子序列;双萤光素酶实验和荧光显微镜检测发现CD31启动子特异性在内皮细胞中表达。结论:CD31启动子的萤光素酶和绿色荧光表达载体构建成功,可作为鉴定内皮细胞的新型技术手段。

Objective:To construct eukaryotic expression vectors with CD31-promoter-promoted green fluorescent protein(copGFP)and luciferase,and detect their activities in endothelial cell lines,HUVEC and BEND3.These vectors can be used as a new tool to identify endothelial cells.Methods:The sequence of CD31promoter was amplified from the genomic DNA library and cloned into pGL4.0and pCDH-copGFP vector respectively.After being verified by restriction enzyme digestion and sequencing,the constructed vectors were transiently transfected into HUVEC,BEND3and MCF-7.Luciferase activity and green fluorescence signal were subsequently detected.Results:Sequencing analysis proved that CD31promoter sequence was successfully cloned into the expression vectors.Luciferase assay and fluorescence signal detection showed that CD31promotor was specifically activated in endothelial cell lines.Conclusion:The eukaryotic expression vectors with CD31-promoter-promoted green fluorescent protein and luciferase were successfully constructed,which can be a new method to identify endothelial cells.