鼠A20基因的腺病毒载体构建及耳蜗毛细胞表达  

Construction of recombinant adenovirus of mouse A20 gene and identification of cochlear hair cells

作　　者：袁伟[1] 张学渊[1] 侯春丽[2] 魏勇[1] 朱楚洪[2] 

机构地区：[1]第三军医大学西南医院耳鼻咽喉科,重庆400038 [2]第三军医大学解剖学教研室,重庆400038

出　　处：《免疫学杂志》2008年第3期337-339,345,共4页Immunological Journal

基　　金：重庆市自然科学基金资助项目

摘　　要：目的构建鼠锌指蛋白A20基因的重组腺病毒载体,并观察其对豚鼠耳蜗毛细胞的感染情况。方法采用基因工程技术,经亚克隆后将A20基因片段克隆至穿梭质粒pAdTrack-CMV上,利用pAdEasy系统进行细菌内同源重组后,脂质体转染HEK293细胞包装、扩增,将重组腺病毒感染耳蜗毛细胞。PCR方法对重组体腺病毒进行鉴定,利用穿梭质粒中带有绿色荧光蛋白GFP报告基因对病毒滴度和感染情况进行监测。结果酶切鉴定及PCR结果表明A20基因重组腺病毒载体构建成功,病毒滴度达5×109pfu/mL,并对耳蜗毛细胞有很强的感染能力。结论应用细菌内同源重组法成功构建了含鼠A20基因的重组腺病毒载体。Objective To construct the recombinant adenovirus of mouse A20 gene and to observe its ability to infect hair cells of guinea pig inner ear. Methods The A20 gene fragment was cloned into the shuttle plasmid pAdTrack-CMV to form the transfer vector by the method of homogenous recombination in bacteria. Then the recombinant adenovirus was transfected into 293 cells using liposome DOTAP. The target gene was detected by polymrase chain reaction （PCR）. The titer and infection rate of the recombinant adenovirus were determined using the green fluorescent protein （GFP） expression in the shuttle plasmid. Results Restriction endonuclease and PCR analyses confirmed that the A20 gene was successfully inserted into the adenovirus vector. The titer of the recombinant adenovirus was 5 × 10^9 pfu/mL. The adenovirus had a strong effect on hair cells of inner ear. Conclusion The recombinant adenovirus containing A20 gene is successfully constructed by the method of homogenous recombination in bacteria.

关 键 词：腺病毒 A20基因 同源重组 

分 类 号：Q78[生物学—分子生物学]