摘要
目的:克隆人早期B细胞因子3(EBF3)基因,构建真核表达载体pEGFP/EBF3,研究人EBF3对肝癌细胞株HepG2细胞周期的影响。方法:提取人胎盘组织总RNA,采用RT-PCR技术克隆人EBF3基因,并将其克隆到真核表达载体pEGFP-N1,重组质粒双酶切鉴定,并进行序列测定。将重组质粒转染HepG2细胞,Western blot确证EBF3-EGFP融合蛋白的表达及亚细胞定位,流式细胞术分析转染细胞周期。结果:成功获得全长人EBF3基因,经双酶切和序列测定鉴定,真核表达质粒pEGFP/EBF3构建正确,将pEGFP/EBF3导入HepG2细胞24小时后,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。Western blot证实转染pEGFP/EBF3后24小时和48小时细胞质和细胞核中均检出相对分子质量(Mr)为87 000的EBF3-EGFP融合蛋白。转染pEGFP/EBF3后48小时和72小时,S期细胞比例均明显高于pEGFP-N1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质粒pEGFP/EBF3,pEGFP/EBF3转染组S期细胞比例高于pEGFP-N1转染组,提示EBF3对人肝癌细胞株HepG2细胞有促进增殖作用。
Objective:To clone the encoding sequence of human EBF3 gene, construct recombinant eukaryotic expression plasmid vector pEGFP/EBF3, and study the effect of EBF3 on HepG2 cell cycling.Methods: Total RNA was isolated from placental tissue.Full-length human EBF3 cDNA was amplified by RT-PCR, cloned into eukaryotic expression plasmid vector pEGFP-N1 and sequenced. The expression and sub-cellular localization of the fusion protein EBF3-EGFP in HepG2 cells were analyzed by Western blot. Cell cycles were analyzed with flow cytometry analysis. Results: Obtained full encoding sequence of early B cell factor 3 was identical with that included in GeneBank, and the eukaryotic expression plasmid vector pEGFP/EBF3 was constructed correctly. 24 h after transfected by pEGFP/EBF3, the fusion protein EBF3- EGFP was observed mainly in the cellular nucleus under the inverted fluorescence microscope. Western blot analysis confirmed that the EBF3- EGFP fusion proteins of Mr 87 0(30 were detected in both cytoplasmic and nuclear protein of the HepG2 transfected by pEGFP/EBF'3 for 24 h or 48 h. Flow cytometry analysis revealed that the percentage of cells in the S phase was markedly increased in HepG2 cells transfected by pEGFP/EBF3 as compared with that in pEGFP-N1 transfected cells. These findings suggested that transfection of EBF3 gene into HepG2 induced cell proliferation by increasing the number of cells from G1 phase to G2 phase. Conclusion: The recombinant eukaryotic expression plasmid vector pEGFP/EBF3 is successfully established. The percentage of ceils in the S phase is markedly increased in pEGFP/EBF3 transfected cells as opposed to pEGFP-N1 transfected cells. It is likely that EBF3 promotes HepG2 ceils proliferation through DNA replication.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2009年第6期491-495,共5页
Chinese Journal of Immunology
基金
江苏省科教兴卫工程医学重点学科基金资助(XK200723)