EBF3与EGFP融合蛋白表达载体的构建及其在HepG2中的表达

Construction of eukaryotic expression vector for EBF3 and EGFP fusion protein and its expression in HepG2 cells

目的:构建早期B细胞因子3(EBF3)与增强型绿色荧光蛋白(EGFP)的融合基因真核表达载体pEGFP/EBF3,使EBF3-EGFP融合蛋白在人肝癌细胞株HepG2中得到表达。方法:采用RT-PCR技术,从人胎盘组织总RNA中逆转录并扩增出EBF3全长编码基因,构建EBF3与EGFP的融合基因真核表达载体pEGFP/EBF3,用脂质体转染技术将pEGFP/EBF3导入HepG2,使EBF3-EGFP融合蛋白得到表达。结果:经转化细菌、抽提质粒、酶切鉴定和DNA序列分析证实,EBF3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/EBF3。将pEGFP/EBF3导入HepG2细胞24h后,在倒置荧光显微镜下可观察到EBF3-EGFP融合蛋白主要表达于核内,转染pEGFP/EBF3和pEG-FP-N1后48h的转染效率分别为52%和59%。Westernblot证实转染pEGFP/EBF3后24h和48h细胞质和细胞核中均检出相对分子质量(Mr)为87000的EBF3-EGFP融合蛋白,转染pEGFP/EBF3后48h和72h,S期细胞比例均明显高于pEGFP-N1转染细胞组和未转染细胞组,表明EBF3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:成功构建了真核表达载体pEGFP/EBF3,并在HepG2细胞中进行了表达,为进一步研究EBF3的功能提供实验基础。

AIM：To construct the eukaryotic expression vector for early human B-cell factor 3（EBF3）fused with the enhanced green fluorescent protein（EGFP）and express the fusion protein EBF3-EGFP in HepG2 cells.METHODS：The intact EBF3 gene was amplified from RNA isolated from the placental tissue by RT-PCR.Then it was inserted into the pEGFP-N1 vector to construct the recombinant eukaryotic expression vector pEGFP/EBF3.The fusion protein EBF3-EGFP was expressed in HepG2 cells by the transfection of pEGFP/EBF3 into the cells.RESULTS：The pEGFP/EBF3 recombinant expression vector was constructed and confirmed by DNA sequencing,enzymatic digestion and PCR identification.24 h after the fusion protein EBF3-EGFP was transfected wiht pEGFP/EBF3,it was observed mainly in the cellular nucleus under the inverted fluorescence microscope.Both pEGFP/EBF3 and pEGFP-N1 were transfected into human HepG2 cells.24 h after the transfection,the proportion of transfection was about 52% and 59%,respectively.Western blot analysis confirmed that the EBF3-EGFP fusion proteins of Mr 87 000 were detected in the cytoplasmic and nuclear protein of HepG2 transfected with pEGFP/EBF3 for 24 h or 48 h.The cell proportion in S phase increased in HepG2 cells transfected with pEGFP/EBF3 in comparison with HepG2 cells transfected with pEGFP-N1 or untransfected.These findings suggested that the transfection of EBF3 gene into HepG2 induced the cell proliferation from G1 phase to G2 phase by increasing the number of cells.CONCLUSION：The construction of pEGFP/EBF3 eukaryotic expression vector and the expression of EBF3-EGFP fusion protein in HepG2 cells lay a foundation for further study of the relationship between EBF3 and its growth and the proliferation of tumor cells.