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【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1.7kb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。
【Objective】 To construct a mouse T-bet recombinant adenovirus vector and provide an effective T-bet biological expression system for the study of T-bet gene in the treatment of bronchial asthma. 【Method】 T-bet cDNA fragment of about 1.7 kb was cut from plasmid T-bet / GFP-RV by restriction endonuclease and ligated with shuttle plasmid pShuttle. The expression of T-bet cDNA was detected by restriction enzyme digestion The recombinant plasmid pAdeno-T-bet was ligated with the Adeno-X adenovirus vector to construct the recombinant vector pAdeno-T-bet. The DNA sequence of the recombinant plasmid was identified with no mismatch insertion and translocation, and transfected into HEK293 cells. After CPE, cells containing virus supernatant Identification of virus DNA from culture medium by PCR. 【Result】 PCR and restriction enzyme digestion confirmed that T-bet cDNA was correctly cloned into the shuttle plasmid pShuttle. The expression cassette with T-bet cDNA was successfully recombined into the E1A deletion region of the adenovirus vector and successfully packaged in HEK293 cells Recombinant adenovirus pAdeno-T-bet. 【Conclusion】 The recombinant adenovirus vector of mouse T-bet gene was successfully constructed in this experiment and the recombinant adenovirus was successfully packaged in HEK293 cells.