携带CUEDC1基因的慢病毒载体的构建及其对MOLT-4细胞株增殖和集落形成能力的影响

Construction of A Lentiviral Vector Carrying CUEDC1 Gene and Its Effect on the Proliferation and Colony-formating Ability of MOLT-4 Cells

目的:构建过表达人CUEDC1的慢病毒载体pCDH-CUEDC1,利用慢病毒介导的感染获得稳定表达重组质粒的MOLT-4细胞,分析过表达CUEDC1对MOLT-4细胞增殖和集落形成能力的影响。方法:利用RT-PCR的方法获得CUEDC1全长片段,将CUEDC1 DNA片段酶切回收后克隆至慢病毒转移质粒pCDH-CMV-MCS-EF1-copGFPT2A-Puro(以下简称pCDH),获得pCDH-CUEDC1慢病毒重组质粒。经三质粒包装系统包装后获得高滴度慢病毒颗粒并感染人白血病细胞系MOLT-4,通过流式细胞仪分选获得稳定表达的白血病细胞系,应用real-time PCR和Western blot方法分别检测稳定转染细胞系的CUEDC1 mRNA和CUEDC1的表达。应用CCK-8法和集落形成试验测定CUEDC1对MOLT-4细胞增殖和集落形成能力的影响。结果:经限制性内切酶检测及基因测序证实成功构建了携带CUEDC1的重组慢病毒载体;病毒感染MOLT-4细胞后经流式细胞仪分选获得GFP阳性细胞。实时定量PCR及Western blot证实,病毒感染后CUEDC1的mRNA和蛋白表达上调;CCK-8法及集落形成试验显示,过表达CUEDC1促进MOLT-4细胞的增殖和集落形成能力,与对照组相比,均有统计学差异(P<0.05)。结论:成功构建了表达CUEDC1的慢病毒载体,CUEDC1可以促进MOLT-4细胞的增殖和集落形成能力。

Objective:To construct a lentiviral vector carrying human CUEDC1 gene,to establish leukemic cell line MOLT-stably expressing recombinant plasmid,to analyze the expression of CUEDC1 in MOLT-4 cells and to investi-gate its effect on the proliferation of MOLT-4 cells.Metliods:The CUEDC1 gene was amplified by RT-PCR,and then was subcloned into the lentiviral vector pCDH to generate a lentiviral vector pCDH-CUEDC1.Recombinant lentivirus was generated by co-transfection of 3 plasmids,and transfected into MOLT-4 cells.The Real-time PCR and Western blot were respectively applied to detect the expression of CUEDC1 mRNA and protein,the CCK-8 and colony formation as-say were used to evaluate the effect of CUEDC1 on proliferation of MOLT-4 cells.Results:The recombinant lentiviral vector pCDH-CUEDC1 had been constructed successfully.After infection of MOLT-cells with the lentivirus,the re-combinant plasmid could stably up-regulate the expression of CUEDC1 and protein.The CCK-8 detection and colony formation assay showed that exogenous CUEDC1 could significantly promote cell growth and the colony formation of MOLT-cells.Conclusion:The recombinant lentiviral vector carrying human CUEDC1 has been successfully construc-ted,exogenous CUEDC1 can significantly promote cell growth and the colony formation of MOLT-4 cells.