核心结合因子α1过表达慢病毒载体的构建及鉴定

Reconstruction and Identification of Recombinant Lentiviral Vector for Human CBFα1/RUNX2

目的构建含人CBFα1/RUNX2基因的过表达慢病毒载体。方法采用PCR技术体外扩增人CBFα1/RUNX2,将扩增产物与慢病毒载体pGC-FU连接,构建重组质粒pGC-FU-hCBFα1/RUNX2,并进行酶切及测序鉴定。鉴定正确的克隆转染293T细胞,经荧光显微镜观察和Western Blot检测hCBFα1/RUNX2基因在293T细胞内的瞬时表达。再利用脂质体转染法将pGC-FU-hCBFα1/RUNX2、pHelper1.0和pHelper2.0三质粒共转染293T细胞,包装产生慢病毒,并通过实时荧光定量PCR检测病毒滴度。结果重组质粒经测序证实,插入片段与人CBFα1/RUNX2基因序列完全一致。荧光显微镜观察以及Western Blot印迹均证实pGC-FU-hCBFα1/RUNX2中携有正确的hCBFα1/RUNX2基因,并能在293T细胞中瞬时表达。包装慢病毒后实时荧光定量PCR检测病毒滴度为(2.00×108)TU/mL。结论成功构建了携带hCBFα1/RUNX2基因的重组慢病毒载体,为进一步研究其在牙周组织再生中的生物学功能奠定了基础。

Objective To construct a recombinant lentiviral vector for human CBFα 1/RUNX2. Methods A lentiviral expression vector for human CBFα 1/RUNX2 was constructed by recombinant DNA technique. The resulting lentiviral vector containing hCBFα1/ HUNX2 was confirmed by PCR and sequencing. The generated recombinant pGC-FU-hCBFα 1/RUNX2 was transfected into 293T cells and the expression of hCBFα1/RUNX2 was detected by Western- blot. Then 293T cells were cotransfected with lentiviral vector pGC-FU-hCBFα1,pHelper 1.0 and pHelper2.0 by Lipofectamine 2000-mediated transfection. The titer of concentrated virus was detected by real-time PCR. Results The sequence analysis of recombinant plasmid pGC-FU-hCBFα 1/RUNX2 showed that the human CBFα 1/ RUNX2 was inserted into lentiviral vector pGC-FU accurately. Human CBFα1/ RUNX2 expression was observed in 293T cells by Western-blot. The titer of concentrated virus was 2.00 ×10^8U/mL. Conclusion The recombinant lentiviral vector pGC-FU-hCBFα 1/RUNX2 can be constructed correcdy and transfected into 293T cells.