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目的构建小鼠Fancd2os基因的真核表达质粒,并分析Fancd2os基因对人胚肾上皮细胞(HEK293T)增殖和凋亡的影响。方法以小鼠睾丸组织DNA为模板扩增Fancd2os基因c DNA全长片段,并克隆至真核表达载体p3×FLAG-CMV-14,构建重组真核表达质粒p3×FLAG-CMV-14-Fancd2os。经Lipofectamine TM 2000将重组真核表达质粒瞬时转染HEK293T细胞,分别采用RT-PCR及Western blot法检测转染细胞中Fancd2os基因m RNA转录及蛋白表达情况;经CCK8法和流式细胞术分别检测过表达Fancd2os对HEK293T细胞增殖能力及紫外线诱导HEK293T细胞凋亡的影响。结果经双酶切及测序鉴定,重组真核表达质粒p3×FLAG-CMV-14-Fancd2os构建正确;转染p3×Flag-CMV-14-Fancd2os的HEK293T细胞可见Fancd2os基因m RNA转录及其蛋白表达;过表达Fancd2os的HEK293T细胞的增殖活性显著下降(P<0.05);过表达Fancd2os可显著促进紫外线诱导的HEK293T细胞凋亡(P<0.01)。结论成功构建了重组真核表达质粒p3×Flag-CMV-14-Fancd2os,过表达Fancd2os可抑制HEK293T细胞的增殖,促进其凋亡。
Objective To construct the eukaryotic expression plasmid of mouse Fancd2os gene and analyze the effect of Fancd2os gene on the proliferation and apoptosis of human embryonic kidney epithelial cells (HEK293T). Methods The full length cDNA fragment of Fancd2os gene was amplified from mouse testis DNA and cloned into eukaryotic expression vector p3 × FLAG-CMV-14 to construct recombinant eukaryotic expression vector p3 × FLAG-CMV-14-Fancd2os. The recombinant eukaryotic expression plasmids were transiently transfected into HEK293T cells by Lipofectamine TM 2000. The m RNA transcription and protein expression of Fancd2os gene were detected by RT-PCR and Western blot respectively. The expression of Fancd2os mRNA and protein was detected by CCK8 and flow cytometry Effects of Fancd2os Overexpression on Proliferation of HEK293T Cells and Apoptosis of HEK293T Cells Induced by Ultraviolet. Results The recombinant eukaryotic expression plasmid p3 × FLAG-CMV-14-Fancd2os was constructed correctly by double enzyme digestion and sequencing. The m RNA transcription and protein expression of Fancd2os gene was observed in HEK293T cells transfected with p3 × Flag-CMV-14-Fancd2os (P <0.01). The proliferation activity of HEK293T cells overexpressing Fancd2os was significantly decreased (P <0.05). Overexpression of Fancd2os significantly promoted the apoptosis of HEK293T cells induced by UV light (P <0.01). Conclusion The recombinant eukaryotic expression vector p3 × Flag-CMV-14-Fancd2os was constructed successfully. Overexpression of Fancd2os can inhibit the proliferation and promote the apoptosis of HEK293T cells.