靶向COX-2基因短发夹RNA重组表达载体的构建及鉴定

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

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-2mRNA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-2mRNA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。

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.