人P115基因重组真核表达质粒的构建及其对肝癌细胞增殖的影响  

Construction of Eukaryotic Expression Vector for Human P115 Gene and Its Effect on Proliferation of Hepatocarcinoma Cells

作　　者：屈玉玲[1] 易永芬[1] 邓玮[1] 文雪[1] 闫田静[1] 

机构地区：[1]重庆医科大学病理学教研室分子医学与肿瘤研究中心,重庆400016

出　　处：《中国生物制品学杂志》2011年第5期517-521,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助项目(30672431)

摘　　要：目的构建人P115基因重组真核表达质粒,并探讨其过表达对人肝癌细胞HepG2增殖活性的影响。方法采用RT-PCR从HepG2细胞中扩增人P115基因,插入pEGFP-N1 Vector(+)载体,构建重组真核表达质粒pEGFP-N1 Vector(+)-USO1,将重组质粒转染至HepG2细胞,免疫荧光及流式细胞术检测转染效率;RT-PCR及Western blot检测转染细胞中P115基因及蛋白的表达;MTT法检测转染细胞的增殖活性、流式细胞术检测细胞周期变化。结果重组真核表达质粒pEGFP-N1 Vec-tor(+)-USO1经酶切鉴定及测序证明构建正确;重组质粒转染HepG2细胞的转染效率达84.83%;重组质粒转染的HepG2细胞中P115基因和蛋白的表达水平及细胞增殖活性均明显高于空载体转染和未转染的细胞;重组质粒转染的细胞G1/G2期比例明显减少,S期比例明显增加。结论已成功构建了人P115基因重组真核表达质粒,其在肝癌细胞中过表达P115可促进肝癌细胞增殖。Objective To construct a eukaryotic expression vector for human P115 gene and investigate the effect of the expression on proliferative activity of human hepatocarcinoma HepG2 cells.Methods Human P115 gene was amplified by RT-PCR from HepG2 cells and inserted into plasmid pEGFP-N1 Vector（＋）.The constructed recombinant plasmid pEGFP-N1 Vector（＋）-USO1 was transfected to HepG2 cells.The transfection efficacy was determined by IFA and flow cytometry.The transfected cells were determined for expressions of P115 mRNA and protein by RT-PCR and Western blot,for proliferative activity by MTT method,and for cell cycle by flow cytometry.Results Restriction analysis and sequencing proved that recombinant plasmid pEGFP-N1 Vector（＋）-USO1 was constructed correctly.The transfection efficacy of HepG2 cells with the recombinant plasmid was 84.83%.The expression levels of P115 gene and protein as well as proliferative activity of HepG2 cells transfected with the recombinant plasmid were significantly higher than those with empty vector and untransfeted.The percentage of transfected cells at G1 / G2 phases decreased significantly,while that at S phase increased significantly.Conclusion A recombinant eukaryotic expression vector for human P115 gene was successfully constructed and over-expressed in hepatocarcinoma cells,and the expressed P115 promoted the proliferation of hepatocarcinoma cells.

关 键 词：肝肿瘤 高尔基体转运蛋白 P115基因 真核细胞 基因表达 细胞增殖 

分 类 号：Q782[生物学—分子生物学] R735.7[医药卫生—肿瘤]