| 禽源U6启动子介导鸡马立克氏病病毒gI、gE基因特异siRNA的筛选及干扰活性鉴定 |
| |
| 引用本文： | 全炎铭,崔红玉,赵妍,赵晓岩,石星明,高宏博,闫帅,张晓艳,王玫,王云峰.禽源U6启动子介导鸡马立克氏病病毒gI、gE基因特异siRNA的筛选及干扰活性鉴定[J].中国预防兽医学报,2011,33(5). |
| |
| 作者姓名： | 全炎铭 崔红玉 赵妍 赵晓岩 石星明 高宏博 闫帅 张晓艳 王玫 王云峰 |
| |
| 作者单位： | 1. 中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/禽传染病研究室,黑龙江,哈尔滨,150001
2. 中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/禽传染病研究室,黑龙江,哈尔滨,150001;东北农业大学动物科技学院,黑龙江,哈尔滨,150030 |
| |
| 基金项目： | 兽医生物技术国家重点实验室基本科研业务费项目 |
| |
| 摘 要： | 为筛选鸡马立克氏病病毒(MDV)gI、gE基因特异性siRNA,本研究以鸡胚成纤维细胞(CEF)的DNA为模板,扩增禽源cU6-3启动子序列,并合成针对gI和gE基因各5个靶位点的10条shRNA序列.通过重叠PCR将cU6-3启动子与shRNA融合扩增制备shRNA表达盒,将制备的shRNA表达盒分别与重组质粒pEGFP-gI或pEGFP-gE共转染CEF细胞,并根据荧光显微镜观察和流式细胞仪检测结果评价siRNA抑制融合荧光蛋白表达的效率.结果表明,gI和gE基因的抑制效率为10%～80%.分别将其中抑制效率最高的一条shRNA插入pGEM-T载体中构建重组表达质粒pcU6-shgI735和pcU6-shgE936,通过不同细胞系稳定干扰试验和抑制病毒复制试验评价其干扰活性.结果显示,pcU6-shgI735和pcU6-shgE936能够明显抑制融合蛋白的表达及MDV在CEF细胞中的增殖,并且禽源U6启动子构建的表达盒在禽源细胞(DF1)中的活性比在哺乳动物细胞(Vero、MDCK)的活性高1.21～1.45倍.本研究鉴定获得了gI和gE基因的特异性siRNA,筛选出能够抑制MDV复制的靶位点,为MDV基因的功能和抗MDV病毒研究奠定了基础.
|
| 关 键 词： | 鸡马立克氏病病毒 gI和gE基因 禽源U6启动子 siRNA |
Selection and evaluation of inhibitory effect of chicken U6 promoter-driven siRNAs specific to gI and gE gene of Marek's disease virus |
| |
| QUAN Yan-ming,CUI Hong-yu,ZHAO Yan,ZHAO Xiao-yan,SHI Xing-ming,GAO Hong-bo,YAN Shuai,ZHANG Xiao-yan,WANG Mei,WANG Yun-feng.Selection and evaluation of inhibitory effect of chicken U6 promoter-driven siRNAs specific to gI and gE gene of Marek's disease virus[J].Chinese Journal of Preventive Veterinary Medicine,2011,33(5). |
| |
| Authors: | QUAN Yan-ming CUI Hong-yu ZHAO Yan ZHAO Xiao-yan SHI Xing-ming GAO Hong-bo YAN Shuai ZHANG Xiao-yan WANG Mei WANG Yun-feng |
| |
| Institution: | QUAN Yan-ming1#,CUI Hong-yu1,2#,ZHAO Yan1,ZHAO Xiao-yan1,SHI Xing-ming1,GAO Hong-bo1,YAN Shuai1,ZHANG Xiao-yan1,WANG Mei1,WANG Yun-feng1 (1. Division of Avian Infection Diseases,State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150001,China,2. College of Animal Medicine,Northeast Agricultural University,Harbin 150030,China) |
| |
| Abstract: | To select and evaluate the siRNA targeting the gI and gE gene of Marek's disease virus (MDV), ten shRNAs were synthesized and fused with chicken U6-3 (cU6) promoter to construct shRNA expression cassettes by overlapping PCR, respectively. The PCR product of the shRNA expression cassette, together with recombinant plasmid pEGFP-gI or pEGFP-gE,were co-transfected into CEF, respectively, and the fluorescence intensity in the transfected cells were monitored by fluorescence microscopy and flow cytometry. The re... |
| |
| Keywords: | Marek's disease virus gI and gE gene chicken U6-3 promoter siRNA |
| 本文献已被 CNKI 万方数据 等数据库收录！ |
|
