环氧化酶2特异性siRNA真核表达质粒的构建与鉴定  

Construction and identification of the eukaryotic expression plasmid for cyclooxygenase-2-specific siRNA

作　　者：王磊[1] 陈卫昌[1] 杨吉成[2] 

机构地区：[1]苏州大学附属第一医院消化病科,215006 [2]苏州大学医学院细胞与分子生物学教研室

出　　处：《江苏医药》2008年第6期611-613,I0002,共4页Jiangsu Medical Journal

基　　金：江苏省135重点医学人才基金项目(RC2007076)

摘　　要：目的构建环氧化酶2(COX-2)特异性小干扰RNA(siRNA)真核表达质粒,研究其对人结肠癌细胞COX-2的阻抑作用。方法设计短发夹结构的COX-2siRNA对应模版DNA序列,退火处理后克隆至pGPH1-GFP-Neo质粒,构建重组质粒pGPH1-GFP-Neo-COX-2。将重组质粒转染人结肠癌HT-29细胞,采用RT-PCR、Westernblot分别从mRNA和蛋白水平检测COX-2表达。结果酶切及测序证实质粒pGPH1-GFP-Neo-COX-2构建成功。转染重组质粒72h后可以显著抑制HT-29细胞COX-2mRNA和蛋白表达(P<0.05)。结论成功构建的COX-2siRNA真核表达质粒pGPH1-GFP-Neo-COX-2可以抑制人结肠癌HT-29细胞COX-2基因表达。Objective To clone the recombinant eukaryotic expression plasmid of specific small interfering RNA （siRNA） against cyclooxygenase-2 （COX-2） gene and to evaluate its activity of inhibiting the COX-2 expression in human colon cancer HT-29 ceils. Methods The COX-2 siRNA template DNA sequence for short hairpin RNA（shRNA） was designed and synthetized. The annealed siRNA template was inserted into pGPH1-GFP-Neo plasmid. The recombinant plasmid （pGPH1- GFP-Neo-COX-2） was transformed into DH5a strain and identified by restrictive enzyme digestion and sequence analysis. The effect of the recombinant plasmid on COX-2 expression of human colon cancer HT-29 ceils was detected by RT-PCR and Western blot. Results It was confirmed by restrictive enzyme digestion and sequence analysis that the recombinant plasmid was cloned and the aim sequence was obtained. COX-2 expression of HT-29 ceils was inhibited at mRNA and protein levels 72 hours after transfected with the recombinant plasmid. Conclusion COX-2 siRNA expression plasmid pGPH1-GFP-Neo-COX-2 successfully constructed can inhibit the expression of COX-2 gene in human colon cancer HT-29 ceils.

关 键 词：环氧化酶2 小干扰核糖核酸 真核表达质粒 结肠癌 

分 类 号：R392[医药卫生—免疫学]