高滴度表达抑制转录因子的重组腺相关病毒的制备  

Construction of recombinant adeno-associated virus vector of highly expressed transcription factor in hibitor, ROG

作　　者：王桂芳[1] 杨丹榕[1] 修清玉[1] 施柯[2] 吴丹[2] 黎怀星[2] 孙树汉[2] 

机构地区：[1]第二军医大学长征医院呼吸内科,上海200003 [2]第二军医大学医学遗传教研室

出　　处：《上海医学》2004年第12期898-901,F005,共5页Shanghai Medical Journal

基　　金：国家自然科学基金资助项目 (3 0 170 40 6)

摘　　要：目的　获得表达小鼠抑制转录因子ROG(repressorofGATA -3)基因 ;构建表达ROG的重组腺相关病毒载体 ,制备腺相关病毒。方法　设计、合成引物 ,通过逆转录聚合酶链反应 (RT- PCR)从CTLL- 2细胞总RNA中扩增ROG基因 ,测序成功后 ,最终连接于pAAV- MCS ,双酶切、PCR鉴定阳性重组质粒 ,并在Hela细胞中表达 ;制备高滴度重组腺相关病毒。结果　成功扩增了小鼠ROG基因 ,构建表达ROG基因的重组腺相关病毒表达载体 ,并成功表达于Hela细胞。结论　本实验构建的小鼠ROG重组腺相关病毒将为哮喘的基因治疗研究奠定物质基础。Objective To obtain a recombinant adeno-associated virus (AAV) vector expressing mouse repressor of GATA-3(ROG) including the preparation. Methods The total RNA was extracted from cultured CTLL-2 cells. The gene sequence coding ROG was amplified by one-step reverse transcription-polymerase chain reaction(RT-PCR). The fragment was inserted into AAV (pAAV-MCS) ultimatly after two cloning vectors to obtain the recombinant pAAV-ROG; The Hela cells were cotransfected with pAAV-RC and pHelper. Results The ROG cDNA by RT-PCR was amplified from the total RNA extracted from cultured CTLL-2 cells. The resultant fragment was cloned into pAAV confirmed by restriction digestion and sequencing; high titer recombinative virus was obtained through cotransfecting the Hela cells. Conclusions The mouse repressor of GATA-3 gene recombinated with AAV expressing plasmid lays down the foundation for gene therapy of bronchchial asthma and other allergic diseases.

关 键 词：重组腺相关病毒 表达载体 小鼠 基因 转录因子 HELA细胞 高滴度 酶切 测序 总RNA 

分 类 号：R346[医药卫生—基础医学]