Construction of small hair RNA eukaryotic expression vectors targeting TGF-β<sub>1</sub> gene and its inhibitory effects on TGF-β<sub>1</sub> gene

YIN Zhi-kang; et al

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Construction of small hair RNA eukaryotic expression vectors targeting TGF-β₁ gene and its inhibitory effects on TGF-β₁ gene

更新时间：2024-01-17

- Construction of small hair RNA eukaryotic expression vectors targeting TGF-β₁ gene and its inhibitory effects on TGF-β₁ gene
- Journal of Chongqing Medical University Vol. 34, Issue 4, Pages: 400-404(2009)
- 作者机构：
  
  1. 重庆医科大学附属第一医院泌尿外科
  2. 重庆医科大学检验系实验诊断教研室
- 作者简介：
- 基金信息：
- DOI：
  CLC： R346;R692
- Published：2009，
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[1]尹志康,夏雨果,吴小候,罗春丽,何云锋,刘川.TGF-β_1基因特异性shRNA真核表达载体的构建及其基因抑制作用[J].重庆医科大学学报,2009,34(04):400-404.

YIN Zhi-kang, et al. Construction of small hair RNA eukaryotic expression vectors targeting TGF-β₁ gene and its inhibitory effects on TGF-β₁ gene. [J]. Journal of Chongqing Medical University 34(4):400-404(2009)
[1]尹志康,夏雨果,吴小候,罗春丽,何云锋,刘川.TGF-β_1基因特异性shRNA真核表达载体的构建及其基因抑制作用[J].重庆医科大学学报,2009,34(04):400-404. DOI：

YIN Zhi-kang, et al. Construction of small hair RNA eukaryotic expression vectors targeting TGF-β₁ gene and its inhibitory effects on TGF-β₁ gene. [J]. Journal of Chongqing Medical University 34(4):400-404(2009) DOI：

摘要

目的:构建针对人TGF-β1基因的shRNA表达载体并评价其在人脐静脉内皮细胞株中对TGF-β1基因的抑制作用

筛选出高效的表达载体以用于后续的RNAi研究。方法:利用生物信息学方法设计shRNA

在人TGF-β1基因的mRNA序列中选择靶序列

设计并合成编码的shRNA的两条寡核苷酸序列

经退火成互补双链

再克隆至PGenesil-1质粒。构建2个表达载体PGenesil-TGF-β1-1（pT11）和PGenesil-TGF-β1-2（pT12）

酶切及测序证实后转染至人脐静脉内皮细胞株（ECV304）中

错构载体（pHK）为阴性对照。转染后采用半定量逆转录-聚合酶链反应（RT-PCR）方法检测TGF-β1mRNA表达水平

酶联免疫吸附试验（ELISA）测定TGF-β1蛋白质的表达。结果:酶切及测序证实表达载体pT11、pT12构建成功

两个表达载体的转染率为38.2%和40.1%

两个表达载体对人脐静脉内皮细胞株TGF-β1基因的转录抑制率为58.1%和60.0%

蛋白表达抑制率为38.9%和44.3%。结论:成功构建的针对人TGF-β1基因的shRNA表达载体对脐静脉内皮细胞株TGF-β1基因的转录有明显的抑制作用

并以表达载体pT12的效果为优

为后续移植肾肾病基因治疗的研究奠定了基础。

Abstract

Objective:To construct short hairpin RNA(shRNA)eukaryotic expression vector targetinghuman TGF-β1 gene

and evaluate its inhibitory effects on TGF-β1 gene in endothelial cell lin(eECV304)of human umbilical veins in order to select a high efficacy vector for subsequent RNAi study.Methods:The oligonucleotides targeting TGF-β1 mRNA were designed and synthesized according to bioinformatics methods.The annealed duplex strand were cloned into PGenesil-1 plasmid.Two recombinant plasmids(pT11 and pT12) were verified by enzyme digestion and sequencing

and transfected into endothelial cell line of human umbilical veins.Vector with scramble sequence was set as control.The mRNA expression was measured by semi-quantitative reverse transcriptase-polymerase chain reaction(RT-PCR)and the protein was measured by ELISA.Results:The shRNA expressing vectors targeting TGF-β1 gene were confirmed by SalI enzyme digestion and sequencing.The transfection rate of pT11 and pT12 in ECV304 were 38.2%and 40.1%

respectively.The inhibitory rate of TGF-β1 mRNA were 58.1%and 60.0%

and the inhibitory rate of TGF-β1 protein were 38.9%and 44.3%.Conclusions:The successfully constructed shRNA eukaryotic expression vectors specific for human TGF-β1 gene have significant inhibitory effects on the expression of TGF-β1 mRNA and protein in endothelial cell line of human umbilical veins

and the pT12 is more effective.

关键词

短发夹RNARNA干扰转化生长因子β1质粒

Keywords

Short hairpin RNARNA interferencetransforming growth factor β1plasmid

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Construction of small hair RNA eukaryotic expression vectors targeting TGF-β1 gene and its inhibitory effects on TGF-β1 gene

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摘要

Abstract

关键词

Keywords

references

Construction of small hair RNA eukaryotic expression vectors targeting TGF-β₁ gene and its inhibitory effects on TGF-β₁ gene