摘要
目的在基因表达和基因治疗研究中,目的基因过度表达或不适当的表达将影响实验结果,实现对目的基因表达时间和表达水平的精确调控是关键问题。文中旨在构建带绿色荧光蛋白(enhanced green fluorecence protein,EGFP)并可经米非司酮诱导表达的重组腺病毒载体,以期用于基因治疗研究。方法酶切含有EGFP基因和启动子以及含有米非司酮调控系统的质粒pRS-17,亚克隆到穿梭质粒PDC313上,筛选阳性克隆,测序鉴定正确,将其与Admax腺病毒包装系统的辅助质粒共转染293细胞后,构建出可调控的重组腺病毒载体,利用报告基因引物对病毒进行鉴定,扩增后用氯化铯密度梯度离心纯化病毒,用最终稀释法测定其滴度,并在SW620细胞中检测其基因调控效果。结果成功地构建了携带有米非司酮调控系统的可调控腺病毒载体Ad-RUEGFP,病毒滴度达到6.6×1010pfu/ml。当给予诱导剂米非司酮后,腺病毒载体可以诱导表达EGFP,两者在一定范围内呈正比,而没有米非司酮时,几乎无报告基因的表达。结论腺病毒Ad-RUEGFP能调控外源基因的表达,为下一步体内基因治疗实验奠定了实验基础。
Objective To construct a mifepristone inducible recombinant adenovirus carrying enhanced green fluorecence protein for future gene therapy in vivo. Methods Full length red fluorescent gene and mifepristone regulator cassette was subcloned into PDC313 shuttle plasmid. The positive clone was identified by sequencing. Recombinant adenovirus were produced after cotransfection of the shutter vector PDC-EGFP and adenovirus DNA helper plasmid pBHGlox△E1,3Cre into 293 cells . The reeombined adenovirus was purified by CsC1 density gradient centrifugation and its titer was determined by end point dilution assay. Fluorescence microscopy and FCM were confirmed by the activation of this regulatable recombinant adenovirus vector in infected cells. Results The adenovi- rus vector containing enhanced green fluorecence protein gene was identify by PCR. The virus titer of Ad-RUEGFP was 6.6 × 10^10 pfu/ml. Without the absence of mifepristone, no significant enhanced green fluorecence protein activation was observed, whereas in the presence of mifepristone activation of the enhanced green fluorecence protein was positive correlation with the inducer under definite range. Conclusion A replication-defective recombinant adenovirus vector containing enhanced green fluorecence protein was successfully constructed,which can be inducible by mifcpristone.
出处
《医学研究生学报》
CAS
2010年第1期43-47,共5页
Journal of Medical Postgraduates
关键词
腺病毒载体
米非司酮
基因调控
绿色荧光蛋白
Recombinant adenovirus
Mifepristone
Gene regulation
Enhanced green fluorecence protein
