小鼠 T-bet基因重组腺病毒载体的构建

[目的] 构建小鼠 T-bet基因重组腺病毒载体,为 T-bet基因在支气管哮喘治疗中的作用研究提供有效的 T-bet生物表达系统.[方法] 采用内切酶从质粒 T-bet/GFP-RV中切获约 1.7 kb的小鼠 T-betcDNA片段,与穿梭质粒 pShuttle连接,再通过稀有酶切位点将含 T-betcDNA的表达盒与 Adeno-X腺病毒载体骨架连接,构建重组载体 pAdeno-T-bet,测序鉴定无错配及插入移位等 DNA顺序改变,并转染 HEK293细胞,出现 CPE后取含病毒上清的细胞培养液抽提病毒 DNA行 PCR鉴定.[结果] PCR及酶切证实: T-betcDNA正确克隆到穿梭质粒 pShuttle中,带 T-betcDNA的表达盒成功重组到腺病毒载体基因组 E1A缺失区,并在 HEK293细胞中成功包装出具有感染活性的重组腺病毒 pAdeno-T-bet.[结论]本实验成功构建了小鼠 T-bet基因重组腺病毒载体,并在 HEK293细胞中成功包装出重组腺病毒.

Construction of Murine T-bet cDNA Recombinant Adenovirus Vector and Its Identification

ObjectiveTo construct a murine T-box expressed in T cells(T-bet) cDNA recombinant adenovirus vector for facilitating the study of gene therapy for bronchial asthma. MethodsAn approximate 1.7 kb murine T-bet cDNA segment was excised from plasmid T-bet/GFP-RV. T-bet cDNA was ligated with pShuttle in unique enzyme site which was connected with adenovirus backbone sequence Adeno-X, named as pAdeno-T-bet. Identifying the desired recombinant adenovirus, it was packaged in HEK293 cells. Supernatant of adenovirus from HEK293 cells which was appeared a visible cytopathic effect (CPE) was collected for extracting DNA and PCR amplification was performed with specific primers. ResultThe recombinant plasmids were identified by PCR amplification and restriction enzyme map. The recombinant adenovirus carrying T-bet gene was identified by PCR amplification. ConclusionThe murine T-bet cDNA recombinant adenovirus vector was successfully constructed in this experiment, by which murine T-bet cDNA recombinant adenovirus was then packaged in HEK293 cells.