细菌内同源重组法构建LEDGFp52基因腺病毒载体及体外表达(英文)  

Construction of recombinant adenoviral vector ca rrying the LEDGFp52 gene by homologous recombination in bacteria and its expression in vitro

作　　者：赵海生[1] 王一[1] 

机构地区：[1]第三军医大学西南医院眼科,中国重庆市400038

出　　处：《国际眼科杂志》2006年第5期979-983,共5页International Eye Science

摘　　要：目的: 构建表达人类晶状体来源的上皮生长因子 p52(LEDGFp52)重组腺病毒载体,检测 LEDGFp52 重组腺病毒能否在真核细胞中有效表达目的基因。方法: 将 LEDGFp52 基因片段亚克隆到腺病毒穿梭质粒pAdTrack- CMV上构建腺病毒穿梭载体 pAdTrack- CMV-LEDGFp52,将腺病毒穿梭载体 pAdTrack- CMV- LEDGFp52 与5 型腺病毒骨架质粒 pAdeasy- 1 共转染 BJ5183 细菌, 经细菌内同源重组产生携带 LEDGFp52 基因的重组腺病毒载体 pAd- LEDGFp52, 将 pAd- LEDGFp52 经脂质体法转化293 细胞包装产生重组腺病毒 Ad- LEDGFp52, 将 Ad-LEDGFp52 体外转染 293 细胞, CPE 法及 westernblot 观察目的基因的表达。结果: 构建了表达 LEDGFp52 基因的重组腺病毒质粒, E.coli 内成功同源重组腺病毒, 在 293 细胞内包装产生重组腺病毒 Ad- LEDGFp52, 滴度可达 5×1012 pfu/L。在真核细胞中有效表达目的基因 LEDGFp52, 出现显著的 CPE 效应。结论: 成功构建表达 LEDGFp52 的重组腺病毒载体, 为进一步进行 LEDGF p52 基因功能的研究提供了实验基础。AIM： To construct recombinant adenoviral vector carrying the LEDGFp52 gene by homologous recombination in bacteria and to detect its expression/n v/fro. METHODS： The LEDGFp52 gene was cloned to adenoviral shuttle plasmid pAdTrack-CMV. Then, the resultant pAd- Track-CMV-LEDGFp52 was cotransfected into B J5 183 baderia with the adenoviral backbone plasmid pAdeasy-l. The adenoviral plasmid carrying LEt）GFp52 was generated with homologous recombination in bacteria, and the adenoviruses were produced in 293 cells. These 293 cells were then infected with adenoviruses, and the expression of LEDGFp52 was detected by CPE （cytopathic effect） and western blot. RESULTS： The titer of Ad-LEDGFp52 adenoviruses was up to 5×10^12 pfu/L after proliferation in 293 cells. LEDGFp52 was expressed efficiently in 293 cells after infection. CONCLUSION： The recombinant adenoviruses vector expressing LEDGFp52 was constructed successfully and can be used in further gene transfection experiments.

关 键 词：LEDGFp52基因 腺病毒载体 体外表达 

分 类 号：R346[医药卫生—基础医学]