Cloning and identification of shRNA recombinant plasmid targeting on COX-2 gene

YANG Yi-ming; LIU Yu; TU Zhi-guang; ZHANG Yu-qian

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Cloning and identification of shRNA recombinant plasmid targeting on COX-2 gene

更新时间：2025-01-15

- Cloning and identification of shRNA recombinant plasmid targeting on COX-2 gene
- Journal of Army Medical University Vol. 29, Issue 6, Pages: 503-506(2007)
- 作者机构：
  
  1. 重庆医科大学医学检验系临床生物化学教研室
  2. 重庆市肿瘤医院临床检验中心
  3. 南京大学生命科学院生物化学系
- 作者简介：
- 基金信息：
- DOI：
  CLC： Q554.6;Q782;Q785
- Published：2007，
  
  Published Online：30 March 2007，
- 稿件说明：
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YANG YI-MING, LIU YU, TU ZHI-GUANG, et al. Cloning and identification of shRNA recombinant plasmid targeting on COX-2 gene. [J]. Journal of army medical university, 2007, 29(6): 503-506.
DOI：

YANG YI-MING, LIU YU, TU ZHI-GUANG, et al. Cloning and identification of shRNA recombinant plasmid targeting on COX-2 gene. [J]. Journal of army medical university, 2007, 29(6): 503-506. DOI：

摘要

目的利用RNA干扰技术，以COX-2为靶基因，构建靶向COX-2基因的短发夹RNA（shRNA）重组表达质粒并进行鉴定分析。方法设计、合成1对COX-2编码基因的反向重复序列

中间间隔9个核苷酸序列，经退火形成互补双链，通过定向克隆至质粒pGenesil-1，构建shRNA真核表达载体。转化JM109大肠杆菌，提取质粒进行酶切鉴定和测序分析；RT-PCR和Western blot检测重组表达质粒对COX-2 mRNA和蛋白表达的抑制效果。结果酶切证实目的DNA定向克隆至载体上，测序分析结果与目的序列相同。RT-PCR和Western blot结果显示，与未转染组比较，pshRNA-COX-2重组表达质粒对β-actin基因无明显影响，而对COX-2有明显抑制作用。COX-2 mRNA和蛋白表达的抑制率分别为66.9％和50.3％。结论成功构建的靶向干扰COX-2基因重组质粒能够显著抑制COX-2基因表达。

Abstract

Objective To clone shRNA (short hairpin RNA) recombinant plasmid targeting on COX-2 gene and analyze the nucleic acid sequence of the recombinant plasmid. Methods One pair of 21 bp reverse repeated sequence targeting on COX-2 mRNA spaced by 9 bp nucleotides were synthesized. The complement double strands was formed by annealing and inserted into plasmid pGenesil-1 to generate eukaryotic expression plasmid. The recombinant plasmid was transformed into Jm109 strain

and the recombinant plasmid extracted was identified by restriction enzyme and sequence analysis. Inhibition effects of COX-2 mRNA and protein were detected by RT-PCR and Western blotting respectively. Results The target DNA was directly cloned to vector and the result was correct by sequence analysis. Compared to untransfected group

recombinant expression plasmid pshRNA-COX-2 resulted in reduction of COX-2 mRNA and protein expression to 69.9% and 50.3% respectively. Conclusion The recombinant plasmid targeting on COX-2 gene was successfully constructed

and it inhibited the expression of COX-2 gene significantly.

关键词

环氧化酶-2短发夹RNA重组表达载体

Keywords

cyclooxygenase-2short hairpin RNArecombinant plasmid

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