2008-3-7 15:47:45

人Sef 基因重组腺病毒载体的构建与鉴定

生物工程学报 Chin J Biotech 2008, February 25; 24(2): 193-197

journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061

Received: May 23, 2007; Accepted: July 3, 2007

Supported by: 985 Program of Tsinghua University, Tsinghua-Yue-Yuen Medical Sciences Fund and grants from the National Natural Science Foundation

of China (No. 30530420, 30470888 and 30470703), 973 Project (Nos. 2001CB510006, 2002CB513007 and 2006CB910100), and Beijing Science

Research Funding (No. H020220020310).

Corresponding author: Zhijie Chang. Tel: +86-10-62785076; Fax: +86-10-62773624; E-mail: zhijiec@tsinghua.edu.cn

清华大学985 项目, 清华大学-裕元医学基金资助项目及国家自然基金项目(Nos. 30530420, 30470888, 30470703), 国家重点基础研究发展规划

项目(973)(Nos. 2001CB510006, 2002CB513007, 2006CB910100), 北京市科学研究基金项目(No. H020220020310)。

研究报告

人Sef 基因重组腺病毒载体的构建与鉴定

李智勇, 任永明, 荣知立, 李颖华, 程龙, 王银银, 常智杰

清华大学医学院, 清华大学生物科学与技术系, 生物膜与膜生物技术国家重点实验室, 北京 100084

摘要: 构建人Sef-L 和Sef-S 基因的复制缺陷型重组腺病毒表达载体, 为研究Sef 的功能和作用机制以及Sef 的基因治疗奠定基础。通过PCR 方法以hSef 的表达质粒为模板扩增得到hSef 的编码序列, 亚克隆到穿梭载体pAdTrack-CMV中, 经测序验证之后, 将穿梭载体使用Pme I 酶切线性化, 然后与腺病毒基因组质粒pAdEasy-1 共转化大肠杆菌BJ5183,得到重组的Ad-hSef-L 和Ad-hSef-S 质粒, 最后将Ad-hSef-L 和Ad-hSef-S 质粒使用Pac I 线性化, 转染到 HEK293 细胞中, 包装收获病毒颗粒, 免疫印迹实验鉴定表达, 荧光素酶报告实验验证其功能。成功构建了人Sef 基因的复制缺陷型重组腺病毒表达载体, 获得了有功能的Ad-hSef-L 和Ad-hSef-S 病毒重组子。

关键词: Sef, 腺病毒, FGF, MAPK, PI3K

Construction and Characterization of hSef Recombinant Adenoviral Vectors

Zhiyong Li, Yongming Ren, Zhili Rong, Yinghua Li, Long Cheng, Yingyin Wang, and Zhijie Chang

School of Medicine, Department of Biological Sciences and Biotechnology, State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing, China

Abstract: Sef (similar expression to fgf genes) was identified as a feedback antagonist of FGF signaling in zerbrafish, mouse and human. To construct recombinant adenoviral vectors expressing hSef-L and hSef-S, the coding sequences of the two isoforms were amplified and ligated into pAdTrack-CMV, forming shuttle vectors pAdTrack-CMV/hSef-L-Myc and pAdTrack-CMV/hSef-S-Myc. After sequence confirmation, these two shuttle vector plasmids were linearized by Pme I and then co-transformed respectively with the adenoviral genome vector pAdEasy-1 into E. coli BJ5183. The successful recombinants were selected by Kanamycin and confirmed by Pac I digestion. The recombinant vectors Ad-hSef-L-Myc and Ad-hSef-S-Myc were finally digested with Pac I and transfected into HEK293 cells to pack into viral particles. The virus were amplified in 293 cells and used to infect MEF cells. Western blotting analysis was used to demonstrate the expression of hSef-L-Myc and hSef-S-Myc proteins. The inhibitory effects of the adenovirus mediated Sef expression on FGF signaling was further evaluated by Elk luciferase reporter assay. Our results indicated the constructed virus could produce effectively the proteins and then inhibit FGF signaling in MEF cells.

Keywords: FGF, MAPK, PI3K, Sef, adenovirus

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