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    吴勇, 刘学刚, 赵武, 徐家丽, 郭嘉诚, 李慧敏, 丁周志, 王亚明, 宋伟. 野生型及c.454C>T突变型人血管内皮生长因子A基因重组真核表达载体的体外表达[J]. 蚌埠医科大学学报, 2015, 40(2): 148-151. DOI: 10.13898/j.cnki.issn.1000-2200.2015.02.003
    引用本文: 吴勇, 刘学刚, 赵武, 徐家丽, 郭嘉诚, 李慧敏, 丁周志, 王亚明, 宋伟. 野生型及c.454C>T突变型人血管内皮生长因子A基因重组真核表达载体的体外表达[J]. 蚌埠医科大学学报, 2015, 40(2): 148-151. DOI: 10.13898/j.cnki.issn.1000-2200.2015.02.003
    shu
    WU Yong, LIU Xue-gang, ZHAO Wu, XU Jia-li, GUO Jia-cheng, LI Hui-min, DING Zhou-zhi, WANG Ya-ming, SONG Wei. The expression of recombinant eukaryotic expression vector of wild type and c.454C>T mutant human VEGFA gene in vitro[J]. Journal of Bengbu Medical University, 2015, 40(2): 148-151. DOI: 10.13898/j.cnki.issn.1000-2200.2015.02.003
    Citation: WU Yong, LIU Xue-gang, ZHAO Wu, XU Jia-li, GUO Jia-cheng, LI Hui-min, DING Zhou-zhi, WANG Ya-ming, SONG Wei. The expression of recombinant eukaryotic expression vector of wild type and c.454C>T mutant human VEGFA gene in vitro[J]. Journal of Bengbu Medical University, 2015, 40(2): 148-151. DOI: 10.13898/j.cnki.issn.1000-2200.2015.02.003
    shu

    野生型及c.454C>T突变型人血管内皮生长因子A基因重组真核表达载体的体外表达

    The expression of recombinant eukaryotic expression vector of wild type and c.454C>T mutant human VEGFA gene in vitro

      摘要
    • 摘要: 目的: 探讨野生型和c.454C>T突变型人血管内皮生长因子A(VEGFA)基因重组真核表达载体(pEGFP-VEGFA)体外表达有无差异。方法: 用脂质体法将野生型和突变型pEGFP-VEGFA转染人胚胎肾细胞(HEK293T),采用荧光显微镜观察重组载体VEGFA和增强型绿色荧光蛋白(EGFP)的融合蛋白的表达,以实时荧光定量PCR法检测VEGFA mRNA的表达。结果: 在经转染的HEK293T细胞内观察到绿色荧光,转染后48 h突变型pEGFP-VEGFA转染组荧光强度低于野生型pEGFP-VEGFA转染组。突变型pEGFP-VEGFA转染组VEGFA mRNA表达明显低于野生型pEGFP-VEGFA转染组(P<0.01)。结论: 在HEK293T细胞中,初步表明VEGFA突变体导致VEGFA mRNA和蛋白的表达水平均下调,为进一步研究VEGFA c.454C>T突变体在先天性左心室流出道病变发生中的作用奠定了基础。

       

      Abstract: Objective: To explore whether there is a difference between the expression of recombinant eukaryotic expression vector of wild type and c.454C>T mutant human VEGFA gene(pEGFP-VEGFA) in vitro.Methods: The wild-type and mutant-type pEGFP-VEGFA were transfected into the HEK293T cells by lipofectin reagents,and the expressions of fusion proteins(EGFP and VEGFA)were observed under the fluorescence microscopy after 24 and 48 h post-transfection.VEGFA mRNA expressions were detected by real-time fluorescence quantitative PCR.Results: Green fluorescences were observed in the HEK293T cells transfected with wild-type and mutant-type pEGFP-VEGFA.The intensity of fluorescence in HEK293T cells transfected with mutant-type pEGFP-VEGFA was lower than that transfected with wide-type pEGFP-VEGFA at 48 h post-transfection.The mRNA level of VEGFA in HEK293T cells transfected with mutant-type pEGFP-VEGFA was significantly lower than that transfected with wide-type pEGFP-VEGFA(P<0.05).Conclusions: The recombinant eukaryotic expression vectors of human wild-type and mutant-type pEGFP-VEGFA have transiently expressed in HEK293T cells.Our findings preliminarily show that VEGFA mutant leads to down-regulation of VEGFA mRNA and protein expression level in HEK293T cells,which lays a foundation to further study the role of VEGFA c.454C>T mutant in the development of congenital left ventricular outflow tract obstruction.

       

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