绿色荧光蛋白与鼠盘状结构域受体2融合基因的构建与表达

目的: 构建以绿色荧光蛋白(GFP)为报告基因的重组表达质粒DDR2/pEGFP-N3 并分析该融合基因在活细胞中的表达.方法: 利用反转录PCR扩增鼠盘状结构域受体2(DDR2)cDNA,并重组于pEGFP-N3载体.脂质体体外瞬时转染培养的COS-7细胞,RT-PCR和Western Blot分析该融合基因的表达.结果: 从NIH3T3细胞中克隆到DDR2基因的cDNA,并成功构建DDR2-EGFP融合表达载体.以该质粒转染细胞48 h后,RT-PCR和Western Blot分析表明该融合基因能够在COS-7细胞中正确表达.结论: DDR2/pEGFP-N3融合基因真核表达载体构建成功.该融合蛋白具有DDR2和EGFP双重特性,为进一步研究DDR2的功能提供了工具.

Construction and expression of mouse discoidin domain receptor 2 fused with enhanced green fluorescent protein gene

AIM: To construct DDR2/EGFP fusion gene in eukaryotic expression vector and to investigate its expression in living cells.METHODS: Mouse discoidin domain receptor 2 (DDR2) was amplified by RT-PCR and was inserted into pEGFP-N3.The recombinant expression plasmid DDR2/pEGFP-N3 was transiently transfected into COS-7 cells.RT-PCR and Western Blot were used to evaluate the expression of the fused gene.RESULTS: The results of RT-PCR and Western Blot showed that DDR2/EGFP fusion protein was properly expressed in COS-7 cells.CONCLUSION: We have successfully constructed DDR2/EGFP fusion gene in eukaryotic expression vector.This fusion protein has the characteristics of mouse DDR2 and EGFP and can be used as a potential tool for further study on DDR2 functions.