小鼠Th1细胞特异性转录因子T-bet基因腺相关病毒载体的构建

Cloning of specific transcription factor T-bet of murine T helper 1 type cells

目的:构建小鼠Th1细胞特异性转录因子T-bet基因的腺相关病毒载体。方法:利用RT-PCR方法,从Balb/C小鼠脾细胞的mRNA中扩增T-bet基因全长cDNA,经测序证实后插入Pzac2.1质粒,用磷酸钙沉淀法,与pAddeltaF6及p5E18质粒共转染293细胞,包装成重组的病毒颗粒,并通过抽提病毒DNA进行PCR扩增鉴定重组病毒的形成,设立eGFP基因为对照。结果:1592bp的小鼠T-bet基因被成功克隆,分析表明,与Genbank中发表的序列相比具有99.8%的同源性。重组质粒Pzac2.1-T-bet的PCR及酶切鉴定表明T-bet基因被定向插入,与pAddeltaF6及p5E18质粒共转染293细胞包装成病毒后,经荧光显微镜和病毒DNA的PCR检测,证实已完成对重组病毒的包装。结论:成功构建了小鼠T-bet基因的腺相关病毒载体rAAV-T-bet,为免疫紊乱性疾病的T-bet基因干预治疗奠定了基础。

Objective： To construct recombinant adeno-associated virus （rAAV） vectors of specific transcription factor T-bet of murine T helper 1 type cells. Methods： Total RNA as well as mRNA were isolated from splenic cells of Balb/c mice and the entire coding cDNA sequence of T-bet was amplified by reverse transcription polymerase chain reaction （RT-PCR）. The T-bet cDNA were transfected into the pGEM-T vector with a T base at 3＇ terminal. The identification was made by means of restriction enzyme analysis, PCR and DNA sequencing. The T-bet of correct sequence was inserted into plasmid pzac2.1. The latter together with the plasmids of pAddehaF6 and p5E18 were transfected into 293 cells by calcium phosphate precipitation. After packing, the rAAV was identified by PCR amplification. Meanwhile, eGFP gene was chosen as control. Results： The murine entire coding cDNA of 1592 bp was amplified from spleen cells. There was 99.8% homogenous nucleotide sequence as compared with standard sequence of T-bet from Genbank. It was shown that T-bet gene was inserted into plasmid pzac2.1 in right direction and formed recombinant plasmid pzac2.1-T-bet： When pzac2.1-T-bet, pAddeltaF6 and pSE18 were all transfected into 293 cells, PCR amplification verified that rAAV-T-bet＇s packing was completed. Conclusion： The rAAV of murine specific transcription factor T-bet is established. It will be the basis for studying immune interference of transferring gene.