靶向新城疫病毒NP基因的shRNA重组腺病毒表达载体的构建及鉴定  

Construction and identification of recombinant adenovirus expressing shRNA targeting NP gene of Newcastle disease virus

作　　者：杨帆[1] 岳华[1] 侯巍[1] 汤承[1] 

机构地区：[1]西南民族大学生命科学与技术学院,四川成都610041

出　　处：《中国预防兽医学报》2010年第1期5-8,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金："十一五"国家科技支撑计划项目(2006BAD06A11);国家科技基础条件平台项目(2005DKA21205-11)

摘　　要：为验证腺病毒载体在鸡胚成纤维细胞(CEF)及鸡胚中递送小干扰RNA的可行性,本实验利用共转染技术将表达针对新城疫病毒(NDV)NP基因shRNA的重组质粒同源重组到pGSadeno腺病毒载体系统,用重组腺病毒感染CEF和鸡胚,通过红荧光报告基因的表达情况和荧光定量PCR检测NP基因mRNA的表达对其进行鉴定,并通过细胞形态观察、血凝价的测定评价其在CEF和鸡胚上对NDV增殖的影响。实验结果显示重组腺病毒能够感染CEF,感染CEF后6h、9h、12h与对照组比较NP基因mRNA的表达量分别降低了3.2倍,25.6倍,2.57倍,并能够推迟NDV致细胞病变效应,抑制NDV在鸡胚上的增殖,延缓鸡胚的死亡。本实验构建的重组腺病毒能够在CEF和鸡胚上递送特异的shRNA,为在CEF中的RNA干涉研究开辟了新的递送途径。A recombinant adenovirus expressing shRNA that targeted the NDV NP gene was constructed, shRNA plasmid was inserted into vector pGSadeno by co-transfection. The effect of recombinant adenovirus on NDV proliferation was evaluated in CEF and chicken embryo by examining cell morphological change, and detecting the NP mRNA expression and by HA assay. The result showed that the expression ofNP gene was down-regulate by 3.2, 25.6, 2.57 times respectively at 6 h, 9 h, 12 b after infec- tion by the adnovirus; the presence of CPE in CEF was postponed and death time of chicken embryo after NDV infection was also delayed. These results demonstrate that the recombinant adenovirus could facilitate specific delivery of shRNA into CEF and chick- en embryo, providing a new delivery vehicle in RNA interference.

关 键 词：短发夹样结构小干扰RNA 腺病毒载体 新城疫病毒 

分 类 号：S852.65[农业科学—基础兽医学]