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目的构建人分化抑制因子3(Id3)与增强型绿色荧光蛋白(EGFP)的融合基因表达载体pEGFP/Id3,使Id3-EGFP融合蛋白在人肺癌细胞A549中得到表达。方法扩增并克隆人Id3cDNA,构建Id3和EGFP的融合基因表达载体pEGFP/Id3。用脂质体转染技术将pEGFP/Id3导入A549细胞,使Id3-EGFP融合蛋白在A549细胞得到表达。结果经转化细菌、抽提质粒、酶切鉴定和DNA序列分析证实,Id3基因已正确插入pEGFP-N1中EGFP基因的上游,获得融合基因表达载体pEGFP/Id3。利用脂质体转染技术将pEGFP和pEGFP/Id3转入A549细胞,24h后,在激光共聚焦荧光显微镜下可观察到表达EGFP的细胞发出绿色荧光,流式细胞术分析表明,转染48h时EGFP阳性表达细胞率最高,分别可达65·40%和60·50%。pEGFP/Id3转染的A549细胞S期细胞和G2期细胞比例均明显高于pEGFP转染细胞组和未转染细胞组,表明Id3基因的导入可诱导细胞从G1期向G2期发展,从而促进细胞增殖。结论真核表达载体pEGFP/Id3的成功构建及其在A549细胞中的表达,为研究Id3的细胞定位表达及Id3在细胞生长调控中的作用奠定了实验基础。
Objective To construct a fusion gene expression vector pEGFP / Id3 with human Id3 and EGFP, and to express Id3-EGFP fusion protein in human lung cancer A549 cells. Methods The human Id3 cDNA was amplified and cloned to construct the fusion gene expression vector pEGFP / Id3 of Id3 and EGFP. PEGFP / Id3 was transfected into A549 cells by lipofection technique, and the Id3-EGFP fusion protein was expressed in A549 cells. Results The transformed bacteria, plasmid extraction, restriction enzyme digestion and DNA sequence analysis confirmed that the Id3 gene was correctly inserted into the upstream of the EGFP gene in pEGFP-N1 and the fusion gene expression vector pEGFP / Id3 was obtained. Transfection of pEGFP and pEGFP / Id3 into A549 cells by lipofectamine was performed. After 24h, EGFP-expressing cells were observed under confocal laser scanning confocal microscope to emit green fluorescence. Flow cytometry analysis showed that after transfection 48h The highest EGFP positive rate was 65.4% and 60.5% respectively. The percentage of S phase cells and G2 phase cells of A549 cells transfected with pEGFP / Id3 was significantly higher than that of pEGFP transfected cells and untransfected cells, indicating that the induction of Id3 gene can induce cells to develop from G1 phase to G2 phase, thus promoting Cell Proliferation. Conclusion The successful construction of eukaryotic expression vector pEGFP / Id3 and its expression in A549 cells lay an experimental foundation for studying the location of Id3 and the role of Id3 in cell growth regulation.