人Notch1受体shRNA重组腺病毒载体的构建及鉴定  

Construction and Identification of Recombinant Adenovirus Vector Expressing the shRNA of Human Notch1 Receptor

作　　者：王晓辉[1,2] 罗芸[2] 吕同德[2] 杨宵鹏[2] 唐瑜[2] 哈小琴[2] 

机构地区：[1]第四军医大学细胞生物学教研室细胞工程研究中心,陕西西安710033 [2]兰州军区兰州总医院医学实验中心甘肃省干细胞与基因药物重点实验室,甘肃兰州730050

出　　处：《现代生物医学进展》2013年第15期2846-2851,共6页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(30772572)

摘　　要：目的:构建人特异性受体Notch1短发夹RNA重组腺病毒载体,探讨其对人脐带间充质干细胞中Notch1表达的影响,以便进一步研究Notch1的生物学功能。方法:设计合成Notch1序列干扰序列,插入到pGenesil-1.1上构建重组质粒,取阳性克隆进行酶切及DNA测序鉴定。通过同源重组,构建含目的基因的重组腺病毒质粒载体pGsadeno-Notch1-shRNA。经Pac I线性化后脂质体介导转染到HEK 293细胞,包装后得到腺病毒颗粒。产生的重组腺病毒体外转染人脐带间充质干细胞,RT-PCR和Western blot检测特异性shRNA对人脐带间充质干细胞中Notch1蛋白在mRNA及蛋白表达水平的抑制效果。结果:经酶切及DNA测序重组腺病毒质粒构建正确。重组腺病毒转染人脐带间充质干细胞3 d后,RT-PCR及Western blot检测,可显著下调细胞内Notch1的转录及蛋白表达水平。对Notch1 mRNA和蛋白表达的抑制率分别为70.31%和86.97%,具有统计学意义(P<0.05)。结论:成功构建了表达人Notch1受体shRNA的重组腺病毒载体,为研究Notch1在肿瘤学中的生物学作用及机制奠定基础。Objective： Construction of recombinant adenovirus vector carrying human specific receptor Notch1 short hairpin RNA and investigate it interfere Notch1 expression in human umbilical cord mesenchymal stem cells（hUCMSCs） for further study of the biological function of Notch1.Methods： The RNA interfering sequence targeting Notch1 gene was designed and synthesized,then inserted into pGenesil-1.1 to construct a recombinant plasmid.The positive plasmid was identified by enzyme digestion and DNA sequencing.Recombinant adenovirus plasmid vector,pGsadeno-Notch1-shRNA,containing the target gene was constructed by homologous recombination,.After linearized by Pac I,pGsadeno-Notch1-shRNA was transfected into HEK 293 cells for packaging and amplification by liposome mediation.Recombinant adenovirus transfected into hUCMSCs in vitro and the interference with the expressions of Notch1 mRNA and its protein were detected by RT-PCR and western blot.Results： The recombinant adenovirus vector,pGsadeno-Notch1-shRNA,had been successfully constructed as verified by enzyme digestion and DNA sequencing analysis.After transfection 3 d,detecting by RT-PCR and western blot,the transcription and expressions of Notch1 in hUCMSCs were significantly decreased.The inhibition ratios of Notch1 shRNA at mRNA and protein levels were 70.31 % and 86.97 % respectively with statistical significance（P0.05）.Conclusion： The recombinant adenovirus rAd5-Notch1-shRNA targeting Notch1 gene has been constructed successfully,which provides a basis for investig ating the biological function and mechanism of Notch1 in the tumor.

关 键 词：NOTCH1 腺病毒载体 RNA干扰 基因治疗 

分 类 号：Q75[生物学—分子生物学] Q78