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

吴勇; 刘学刚; 赵武; 徐家丽; 郭嘉诚; 李慧敏; 丁周志; 王亚明; 宋伟

doi:10.13898/j.cnki.issn.1000-2200.2015.02.003

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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

吴勇 , 刘学刚 , 赵武 , 徐家丽 , 郭嘉诚 , 李慧敏 , 丁周志 , 王亚明 , 宋伟

文章导航 > 蚌埠医学院学报 > 2015, 40 > 2: (148-151)

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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

1. 蚌埠医学院第一附属医院儿科, 安徽蚌埠 233004;
2. 蚌埠医学院第一附属医院心胸外科, 安徽蚌埠 233004;
3. 蚌埠医学院第一附属医院超声心动图室, 安徽蚌埠 233004

作者简介: 吴勇(1981-),男,硕士研究生.

基金项目:
安徽省自然科学基金面上项目(11040606M198)
中图分类号: Q71

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

1. Department of Pediatrics, The First Affiliated Hospital of Bengbu Medical College, Bengbu Anhui 233004;
2. Department of Cardiothoracic Surgery, The First Affiliated Hospital of Bengbu Medical College, Bengbu Anhui 233004;
3. Department of Echocardiography, The First Affiliated Hospital of Bengbu Medical College, Bengbu Anhui 233004, China
CLC number: Q71

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。
- 血管内皮生长因子A /
- 突变 /
- 遗传载体 /
- 实时荧光定量聚合酶链反应 /
- 左心室流出道梗阻
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.
- vascular endothelial growth factor A /
- mutation /
- genetic vectors /
- real time fluorescence quantitative PCR /
- left ventricular outflow tract obstruction

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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

作者简介: 吴勇(1981-),男,硕士研究生.

1. 蚌埠医学院第一附属医院儿科, 安徽蚌埠 233004;
2. 蚌埠医学院第一附属医院心胸外科, 安徽蚌埠 233004;
3. 蚌埠医学院第一附属医院超声心动图室, 安徽蚌埠 233004

收稿日期: 2013-08-31

基金项目: 安徽省自然科学基金面上项目(11040606M198)

关键词:

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。

English Abstract

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

1. Department of Pediatrics, The First Affiliated Hospital of Bengbu Medical College, Bengbu Anhui 233004;
2. Department of Cardiothoracic Surgery, The First Affiliated Hospital of Bengbu Medical College, Bengbu Anhui 233004;
3. Department of Echocardiography, The First Affiliated Hospital of Bengbu Medical College, Bengbu Anhui 233004, China

Received Date: 2013-08-31

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.