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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 recombinant eukaryotic expression plasmid of human P115 gene and investigate its effect on the proliferation of HepG2 cells. Methods Human P115 gene was amplified from HepG2 cells by RT-PCR and inserted into pEGFP-N1 Vector (+) vector to construct recombinant eukaryotic expression vector pEGFP-N1 Vector (+) - USO1. The recombinant plasmid was transfected into HepG2 cells, The transfection efficiency was detected by immunofluorescence and flow cytometry. The expression of P115 gene and protein in transfected cells was detected by RT-PCR and Western blot. The proliferation activity of transfected cells was detected by MTT assay. The cell cycle was detected by flow cytometry. Results The recombinant plasmid pEGFP-N1 Vec-tor (+) - USO1 was confirmed by restriction enzyme digestion and sequencing. The recombinant plasmid was transfected into HepG2 cells with a transfection efficiency of 84.83%. The recombinant plasmid transfected HepG2 cells transfected with p115 The expression level of gene and protein and cell proliferation activity were significantly higher than those of transfected and untransfected cells. The percentage of G1 / G2 phase cells transfected with recombinant plasmid was significantly decreased, and the proportion of S phase was significantly increased. Conclusion Recombinant eukaryotic expression plasmid of human P115 gene has been successfully constructed and its overexpression of P115 in hepatoma cells can promote the proliferation of hepatoma cells.