Id3-EGFP融合基因表达载体的构建及其在人肺癌细胞A549中的表达

Construction of Id3-EGFP eukaryotic expression vector and its expression in A549 cells

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 eukaryotic expression vector for human differentiation inhibitor3 （Id3） fused with enhanced green fluorescent protein （EGFP） and express the fusion protein Id3-EGFP in A549 cells. Methods The human Id3 cDNA was amplified and inserted into the pEGFP-N1 vector to construct the recombinant eukaryotic expression vector pEGFP/Id3. The fusion protein Id3-EGFP was expressed in A549 cells by transfection of Id3/pEGFP into the cells. Results The pEGFP/Id3 recombinant expression vector was constructed and confirmed by DNA sequencing, enzymatic digestion and PCR identification. Both pEGFP-N1 and pEGFP/Id3 were transfected into human A549 cells by LipofectamineTM 2000. Flow cytometry analysis revealed that 48 h after transfection, the proportions of EGFP ＋ cells of pEGFP-N1 and pEGFP/Id3 transfected cells were about 65.40% and 60. 50% respectively. The cell proportions in S phase and G2 phase increased in A549 transfected with Id3/pEGFP in comparison with A549 cells transfected with pEGFG-N1 or no transfected. These findings suggested that transfection of Id3 gene into A549 induced cell proliferation by increasing the number of cells from G1 phase to G2 phases. Conclusion The construction of pEGFP/Id3 eukaryotic expression vector and expression of Id3-EGFP fusion protein in A549 cells provide a foundation for the further study of the relationship between Id3 and growth and proliferation of tumor cells.