Cloning and identification the shRNA expression plasmid for Ezrin gene

WANG Zhong-liang; et al

doi:10.13406/j.cnki.cyxb.2010.10.001

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Cloning and identification the shRNA expression plasmid for Ezrin gene

更新时间：2024-01-17

- Cloning and identification the shRNA expression plasmid for Ezrin gene
- Journal of Chongqing Medical University Vol. 35, Issue 10, Pages: 1524-1527(2010)
- 作者机构：
  
  1. 重庆医科大学附属儿童医院骨科
  2. 重庆医科大学附属儿童医院泌尿外科
  3. 儿童发育疾病研究省部共建教育部重点实验室
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.2010.10.001
  CLC： Q782
- Published：2010，
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[1]王忠良,李旭良,李明,张德文,刘传康,覃佳强,刘官倍.Ezrin基因shRNA表达载体的构建及鉴定[J].重庆医科大学学报,2010,35(10):1524-1527.

WANG Zhong-liang, et al. Cloning and identification the shRNA expression plasmid for Ezrin gene. [J]. Journal of Chongqing Medical University 35(10):1524-1527(2010)
[1]王忠良,李旭良,李明,张德文,刘传康,覃佳强,刘官倍.Ezrin基因shRNA表达载体的构建及鉴定[J].重庆医科大学学报,2010,35(10):1524-1527. DOI： 10.13406/j.cnki.cyxb.2010.10.001.

WANG Zhong-liang, et al. Cloning and identification the shRNA expression plasmid for Ezrin gene. [J]. Journal of Chongqing Medical University 35(10):1524-1527(2010) DOI： 10.13406/j.cnki.cyxb.2010.10.001.

摘要

目的:设计并构建能有效下调Ezrin蛋白表达的短发夹状RNA（Small hairpin RNAs

hRNA）表达载体

探讨载体介导的RNA干扰技术用于预防性治疗骨肉瘤肺转移的可行性。方法:依据小干扰RNA（Small interfering RNAs

iRNA）靶序列的设计原则

以Ezrin蛋白编码基因Villin2为靶基因

合成两对编码短发卡结构的两条DNA序列

经退火合成DNA双链

再克隆至shRNA表达载体pGenesil-1质粒中

转化后进行酶切鉴定和DNA序列测定。将构建的载体用脂质体介导转染大鼠骨肉瘤UMR106细胞株

采用RT-PCR和Western blot法检测细胞Ezrin蛋白在mRNA和蛋白水平的表达。结果:成功构建2个针对Ezrin蛋白的shRNA表达载体;其转染大鼠骨肉瘤UMR106细胞后

细胞Ezrin蛋白表达在mRNA及蛋白水平上的表达均显著下降。结论:Ezrin蛋白的shRNA表达载体能显著抑制大鼠骨肉瘤UMR106细胞Ezrin蛋白的表达

本研究为预防性治疗骨肉瘤肺转移的实验奠定基础。

Abstract

Objective:To construct small hairpin RNA (shRNA)expression plasmids which can effectively knock down Ezrin protein expression

and to explore carrier-mediated RNA interference technology for the preventive treatment of osteosarcoma lung metastases. Methods:According to the criteria of designing small hairpin RNA(siRNA)

two villin2(coding gene of Ezrin protein)shRNA template DNA sequences were designed and synthesized. The annealed shRNA template was inserted into expression vector pGenesil-1. The positive plasmids were identified by enzyme digestion and DNA sequencing. Then the plasmids were transfected into rat osteosarcoma UMR106 cells with liposome mediation

and the expressions of Ezrin mRNA and its protein were detected by RT-PCR and Western blot. Results:Two shRNA expression plasmids for Ezrin were constructed successfully. After the transfection

the Ezrin expressions in rat osteosarcoma UMR106 cells were significantly decreased in both mRNA and protein levels. Conclusion:The shRNA expression plasmid for Ezrin can significantly inhibit Ezrin protein expression in rat osteosarcoma UMR106 cells

this studyset up a foundation for experiments of prophylactic treatment of osteosarcoma pulmonary metastasis.

关键词

Ezrin蛋白RNA干扰骨肉瘤肺转移表达载体

Keywords

EzrinRNAinterferenceosteosarcomaLungmetastasisExpression plasmid

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