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目的构建携带FLAG-3NLS-FRB*融合基因的嵌合腺病毒,并检测其在AD-293细胞中的表达。方法将FLAG标记的3段核定位信号(FLAG-3NLS)与突变修饰的雷帕霉素靶FRB结构域(FRB*)基因通过重叠PCR技术拼接成FLAG-3NLS-FRB*,定向克隆至腺病毒穿梭载体pAdTrack-CMV中,测序鉴定后,与腺病毒骨架质粒pAd5F35在大肠杆菌BJ5183中同源重组,获得嵌合腺病毒Ad5F35-FLAG-3NLS-FRB*,感染AD-293细胞进行包装、扩增。采用基因转移单位(Gene transfer unit,GTU)法测定病毒滴度;RT-PCR及Western blot检测FLAG-3NLS-FRB*在AD-293细胞中的转录及表达。结果腺病毒穿梭质粒pAdTrack-CMV-FLAG-3NLS-FRB*经PCR、双酶切及测序鉴定证明构建正确;FLAG-3NLS-FRB*正确克隆至腺病毒骨架质粒中;在AD-293细胞中包装、扩增获得了高滴度的嵌合腺病毒Ad5F35-FLAG-3NLS-FRB*,病毒滴度为2.0×1013GTU/ml;嵌合腺病毒感染AD-293细胞后36 h,在细胞中可检测到FLAG-3NLS-FRB*基因的转录和蛋白的表达。结论成功构建了高滴度的嵌合腺病毒Ad5F35-FLAG-3NLS-FRB*,并在AD-293细胞中成功表达了FLAG-3NLS-FRB*融合蛋白。
Objective To construct a chimeric adenovirus carrying FLAG-3NLS-FRB * fusion gene and test its expression in AD-293 cells. METHODS FLAG-3NLS-FRB * was cloned by FLAG-3NLS-FRB * by FLAG-tagged 3-locus nuclear localization signal (FLAG-3NLS) and mutant modified rapamycin target FRB domain (FRB * The shuttle vector pAdTrack-CMV was sequenced and homologously recombined with the adenoviral backbone plasmid pAd5F35 in E. coli BJ5183 to obtain a chimeric adenovirus Ad5F35-FLAG-3NLS-FRB *. AD-293 cells were infected and packaged, . The virus titer was determined by gene transfer unit (GTU) assay. The transcription and expression of FLAG-3NLS-FRB * in AD-293 cells were detected by RT-PCR and Western blot. Results The adenovirus shuttle plasmid pAdTrack-CMV-FLAG-3NLS-FRB * was confirmed by PCR, restriction endonuclease and sequencing. The correct construction of FLAG-3NLS-FRB * was cloned into adenovirus backbone plasmid. , The titer of the Ad5F35-FLAG-3NLS-FRB * chimeric adenovirus was 2.0 × 1013GTU / ml. After 36 h, the chimeric adenovirus infected AD-293 cells could be detected in the cells Transcription and protein expression to the FLAG-3 NLS-FRB * gene. Conclusion The high titer recombinant adenovirus Ad5F35-FLAG-3NLS-FRB * was successfully constructed and the FLAG-3NLS-FRB * fusion protein was successfully expressed in AD-293 cells.