携带T-bet基因的重组腺病毒载体的构建  

Construction of Recombinant Adenovirus Vector containing T-bet Gene

作　　者：孙克巍[1] 朱学伟[1] 朱武飞[2] 朱冬冬[1] 董震[1] 

机构地区：[1]吉林大学中日联谊医院耳鼻咽喉-头颈外科 [2]吉林大学基础医学院病源免疫细胞遗传学实验中心

出　　处：《中国实验诊断学》2008年第3期293-295,共3页Chinese Journal of Laboratory Diagnosis

基　　金：国家自然科学基金资助项目(30772409)

摘　　要：目的包装携带人T细胞转录因子T-bet基因的重组腺病毒,为研究T-bet在人鼻黏膜中作用的分子机制奠定基础。方法采用RT-PCR方法从健康人PBMC中扩增出转录因子T-bet的cDNA序列,构建重组穿梭载体pDC316-T-bet;采用阳离子脂质体介导将重组穿梭载体pDC316 T-bet与pBHGloxΔE1,3C re质粒共转染293细胞,包装出携带T-bet基因的重组腺病毒rAdT-bet,空斑纯化,PCR鉴定阳性rAdT-bet。结果以空斑纯化后的rAdT-bet为模板,作PCR均扩增出含1633 bp的片断,阳性率达100%,证实T-bet稳定整合于重组腺病毒。结论利用AdMaxTM腺病毒包装系统成功包装了rAdT-bet,为进一步研究T-bet基因在鼻黏膜中作用的分子机制和探讨变应性鼻炎治疗新手段均具有重要意义。Objective To study the critical role of T-bet in human nasal mucosa,the recombinant adenovirus containing T-bet gene was constructed.Methods T-bet gene was amplified from health individuals PBMC using reverse transcription-polymerase chain reaction （ RT-PCR）, and constructed into recombination shuttle vector pDC316T- bet. After cotransfection of the shuttle vector pDC316- T-bet and adenovirus DNA helper plasmid pBHGlox△E1,3Cre into 293 cells,the recombinant adenovirus containing T-bet gene was obtained through homologous recombination and observation of remarkably eytopathie effect. Results High titers of recombinant adenovirus designated as rAd-T-bet were prepared after PCR identification and virus plaque purification. Conelusion Using AdMaxTM adenovirus system, we constructed rAd-T-bet. This work will provide a good foundation for studing the role of T-bet gene in nasa/mucosa immune and for approaching the new therapy on Allergic Rhinitis.

关 键 词：转录因子T-BET 重组腺病毒 变应性鼻炎 

分 类 号：R346[医药卫生—基础医学]