动脉内皮细胞中TIE2启动CREG真核表达质粒pTIE2-CREG-EGFP-N1的表达

背景：血管内皮发生的分子机制是细胞及基因替代治疗的首要前提。 目的：构建一种通过血管内皮细胞特异蛋白TIE2启动子来启动表达的E1A激活基因阻遏子(cellular repressor of E1A-stimulated genes, CREG)和增强绿色荧光蛋白(enhanced green fluorescence protein, EGFP)融合的真核表达质粒。 方法：根据GenBank中公布的TIE2基因的启动子序列,人工合成TIE2启动子DNA序列,经AseⅠ和NheⅠ双酶切后,亚克隆入表达质粒pEGFP-N1中构建pTIE2-EGFP-N1。同时,用BamHⅠ和EcoRⅠ 双酶切pcDNA3.1 myc-His/hCREG质粒得到CREG基因,亚克隆入质粒pTIE2-EGFP-N1中构建pTIE2-CREG-EGFP-N1,酶切鉴定。应用脂质体法将该质粒转染至体外培养的小鼠动脉内皮细胞,荧光显微镜下观察EGFP的表达;Western blot检测CREG蛋白的表达。 结果与结论：经酶切鉴定证实实验构建的pTIE2-CREG-EGFP-N1质粒正确;体外转染48 h,荧光显微镜下可见EGFP的表达,Western blot检测到CREG蛋白的表达。说明实验成功构建了pTIE2-CREG-EGFP-N1重组质粒,其可携带目的基因在小鼠动脉内皮细胞中有效表达。

Construction of pTIE2-CREG-EGFP-N1 eukaryotic expression plasmid and its expression in mouse artery endothelial cells

BACKGROUND:The molecular mechanism of blood vessel endothelium formation is the primary premise for cellular and gene replacement therapy. OBJECTIVE:To construct an eukaryotic expression plasmid for cellular repressor of E1A-stimulated genes (CREG) driven by an endothelial cell specific promoter TIE2 and reported by enhanced green fluorescence protein (EGFP), and then to observe its expression in mouse artery endothelial cells in vitro. METHODS:TIE2 promoter DNA sequence was synthesized according to GenBank, and was attached with AseⅠ and NheⅠrestriction enzyme recognition sites at both ends, respectively. The TIE2 promoter sequence was released from pUC57-TIE2 plasmid by AseⅠ and NheⅠ digestion enzymes, and then was inserted into pEGFP-N1 plasmid to form pTIE2-EGFP-N1 recombinant plasmid. CREG gene fragment was released from pcDNA3.1 myc-His/hCREG plasmid by BamHⅠ and EcoRⅠdigestion enzymes, and then was subcloned into pTIE2-EGFP-N1 plasmid in order to construct pTIE2-CREG-EGFP-N1 plasmid. The recombinant pTIE2-CREG-EGFP-N1 plasmid was then transfected into mouse artery endothelial cells by liposome. The EGFP expression was observed by fluorescence microscopy and the CREG expression was detected by western blot analysis. RESULTS AND CONCLUSION:Identification of pTIE2-CREG-EGFP-N1 by enzyme digestion and sequencing analysis showed that lengths of the target genes which were inserted into the recombinant plasmid were correct, and that the recombinant plasmid was transfected into mouse artery endothelial cells successfully. The strong expression of EGFP was observed by fluorescence microscopy, and the expression of CREG protein was detected by western blot analyis. The results showed that the pTIE2-CREG-EGFP-N1 eukaryotic expression plasmid was successfully constructed and the recombinant vector provides a practical tool in investigating the function and regulation of CREG in the development of cardiovascular diseases and also in producing transgenic mice with endothelial cell specific over-expression of CREG.