Smad4/DPC4基因小发夹RNA质粒表达载体的构建和鉴定  

Construction and identification of the small hairpin RNA expression vector of Smad4/DPC4 gene

作　　者：季国忠[1] 张发明[2] 黄曙[1] 缪林[1] 刘政[1] 喻容彬[3] 王学浩[4] 

机构地区：[1]南京医科大学第二附属医院消化科,江苏南京210011 [2]第四军医大学西京医院解放军消化研究所,陕西西安710033 [3]南京医科大学公共卫生学院,江苏南京210029 [4]南京医科大学第一附属医院肝胆外科,江苏南京210029

出　　处：《医学研究生学报》2006年第11期973-975,979,I0011,共5页Journal of Medical Postgraduates

基　　金：江苏省自然科学基金资助项目(批准号:BK2001168);南京医科大学科技创新基金资助项目(批准号:CX2004004)

摘　　要：目的:构建Smad4基因小发夹RNA(shRNA)表达载体。方法:设计、合成3对编码Smad4基因shRNA的寡核苷酸序列,并分别将其克隆入pGCsi-H1/Neo/GFP质粒,酶切、测序鉴定,脂质体法转染293细胞。实时荧光定量PCR检测RNA干扰(RNA i)的抑制效果。结果:在经HindⅢ和BamHⅠ双酶切分别鉴定三种重组载体后,DNA测序证实小干扰RNA(siRNA)序列正确,并被准确克隆入载体。实时荧光定量PCR检测结果表明三种重组载体psiSm ad4-1、psiSm ad4-2和psiSm ad4-3载体,分别对293细胞中Smad4基因mRNA表达的特异性抑制率为39.00%、8.80%和73.80%。结论:成功构建Sm ad 4 pGCsi-H1/Neo/GFP/shRNA质粒,为后期研究Smad4基因在肿瘤细胞中的作用机制和基因治疗打下基础。Objective：To construct and identify the small hairpin RNA （shRNA） expression vector for Smad4/DPC4 gene. Methods：Three shRNA sequences targeting Smad4 were designed by software and cloned into the expression vector pGCsi-H1/Neo/GFP. DNA sequencing was used to confirm that the plasmids were constructed correctly. The constructed plasmids with different shRNA and the control plasmid were transiently transfected into the 293 cell line cells. Fluorescene quantitative PCR was used to detect the efficiency of RNA interference against Smad 4. Results：Double enzyme （Hind Ⅲ and BamH Ⅰ ） digestion analysis and DNA sequencing confirmed that Samd 4 specific shRNA expression vectors were constructed successfully. Fluorescene quantitative PCR showed that the inhibition rates of Smad4 mRNA expression in cells transfected with shRNA expression vector psiSmad 4-1, psiSmad 4-2 and psiSmad4-3 were 39.00% , 8.80% and 73.80% , respectively, which indicated Smad4 mRNA expression was specifically inhibited. Conclusion： Smad 4 pGCsi-H1/Neo/GFP/shRNA plasmid was constructed successfully, which may provide a novel applicable strategy for gene therapy and study of its role in carcinogenesis.

关 键 词：RNA干扰 SMAD4 小发夹RNA 293细胞 肿瘤 

分 类 号：Q782[生物学—分子生物学]