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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2010, Vol. 30 Issue (01): 1-6    
研究报告     
腺病毒介导的人巨细胞病毒UL49基因小鼠模型的建立
杨丹,崔延伟,李弘剑,李月琴,周琪,曾志锋,张欣,杨光,周天鸿**
暨南大学基因工程药物国家工程研究中心 暨南大学生命科学技术学院 广州 510632
Recombinant Adenovirus Vector Mediated Expression of HCMV UL49 Gene in Mice
YANG Dan,CUI Yan-wei,LI Hong-jian,LI Yue-qin,ZHOU Qi,ZENG Zhi-feng,ZHANG Xin,YANG Guang,ZHOU Tian-hong
National Engineering Research Center of Genetic Medicine, College of Life Science and Technology, Jinan University, Guangzhou 510632, China
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摘要：

建立表达HCMV UL49 基因的转基因小鼠，为抗病毒药物研究提供有效的实验动物模型。本实验将UL49-GFP基因插入腺病毒穿梭质粒pDC316中,构建重组质粒pDC316-UL49-GFP，与腺病毒骨架质粒pBHGloxΔE1,3Cre 通过脂质体介导共转染293 细胞，重组产生腺病毒Ad-UL49-GFP, 经PCR和Western Blot鉴定正确后，大量扩增、纯化，制备高滴度重组腺病毒。纯化腺病毒经尾静脉注射感染小鼠，通过荧光定量PCR 和Western blot 方法，检测UL49 基因在小鼠体内组织分布和表达时相。结果显示UL49基因在小鼠的心、肝、脾、肺、肾组织均有表达，并且表达量由高到低顺序依次是：肝、脾、肾、心、肺，在腺病毒感染第3天在各靶器官表达水平较高，此后逐渐下降，第14天时仅存在肝和脾中。表明表达UL49基因的小鼠模型构建成功。小鼠模型的成功建立为下一步筛选以UL49基因为靶的抗病毒药物奠定了基础。
关键词： 人类巨细胞病毒;  UL49 基因;  腺病毒载体;  动物模型    
Abstract:

Human cytomegalovirus (HCMV) is extremely species specific and does not replicate in experimental animal tissues.To overcome the problem and establish suitable animal models for studying antiviral strategies,  the expression of HCMV UL49 gene was explored in mice. UL49-GFP gene was subcloned into the adenovirus shuttle plasmid pDC316, the products(pDC316-UL49-GFP)were co-transfected with helper plasmid-pBHGloxΔE1,3Cre into HEK293 cell lines by liposome reagent, recombinant adenovirus(Ad-UL49-GFP) was generated and confirmed by PCR and Western blot. Ad-UL49-GFP was propagated in 293 cells and purified. The titer of viral stocks was determined by end-point dilution assay. The purified adenoviruses were delivered into mice via the tail vein injection. Fluorescence quantitative PCR and Western blot experiments were used to examine the tissue distribution and duration of UL49 gene expression. The results showed that the recombinant adenovirus were present in vivo. The expression level in tissues arranged in descending order was liver, spleen, kidney, heart and lung . 3 days after injection, the liver, spleen, kidney, heart and lung expressed protein UL49 in high lever and then declined gradually. 14 days after injection, UL49 protein expression was disappear in some organs except liver and spleen. In conclusion, transgene animal model carrying UL49 gene was successfully established. Therefore, the system may be suitable for selecting anti-HCMV drugs targeting UL49 gene.
Key words: Human cytomegalovirus    UL49 gene    adenovirus vectors    animal model
收稿日期: 2009-10-12 出版日期: 2010-01-27
基金资助:

国家自然科学基金（90608024,30370776）、广东省科技计划（2006B35502002）、广东省自然科学基金(36703)、中国博士后科学基金（20080430845）资助项目
通讯作者: 周天鸿     E-mail: tzth@jnu.edu.cn
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杨丹
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引用本文:

杨丹 崔延伟 李弘剑 李月琴 周琪 曾志锋 张欣 杨光 周天鸿. 腺病毒介导的人巨细胞病毒UL49基因小鼠模型的建立[J]. 中国生物工程杂志, 2010, 30(01): 1-6.

YANG Dan, CUI Yan-Wei, LI Hong-Jian, LI Ru-Qin, ZHOU Qi, CENG Zhi-Feng, ZHANG Xin, YANG Guang, ZHOU Tian-Hong. Recombinant Adenovirus Vector Mediated Expression of HCMV UL49 Gene in Mice. China Biotechnology, 2010, 30(01): 1-6.

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https://manu60.magtech.com.cn/biotech/CN/    或    https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I01/1

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