NK4基因重组慢病毒载体的构建及在肝癌细胞中的表达

摘要

目的 构建共表达人NK4基因和增强型绿色荧光蛋白(EGFP)基因的重组慢病毒载体,转染人高侵袭转移肝癌细胞LM3(HCCLM3),观察其转染效率及表达情况.方法 采用DNA重组技术,将NK4基因克隆至带EGFP的慢病毒表达载体pLenti6.3-内部核糖体进入位点序列(IRES)-EGFP中,并用脂质体介导法将慢病毒包装系统和带NK4基因的质粒pLemi6.3-NK4-IRES-EGFP共转染293T细胞,包装慢病毒并测定滴度.以不同感染复数(MOI)的重组慢病毒感染293T细胞,筛选最适MOI,以最适MOI重组慢病毒感染HCCLM3细胞,观察转染效率.Western blot法测定细胞中NK4蛋白表达水平.结果 成功构建共表达NK4基因和EGFP基因的慢病毒载体pLenti6.3 -NK4 -IRES-EGFP,并对其包装、纯化及浓缩后测定病毒滴度为1.08×108 TU/mL.重组慢病毒感染HCCLM3细胞筛选其最适MOI为7.转染后HCCLM3细胞中可见明显的NK4蛋白表达,而未转染细胞中无NK4蛋白的表达.结论 成功构建了NK4基因的重组慢病毒载体,有效地转染HCCLM3细胞后可高表达NK4蛋白.%Objective To study the transfection and expression level of human NK4 gene in HCCLM3 by constructing NK4 and EGFP fuse gene of recombinant lentiviral vector. Methods The NK4 gene was cloned to lentiviral expression vector with EGFP[pLenti6.3-IRES-EGFP]by recombining DNA technology .The 293T cells were cotransfected with lentiviral packaged systems and NK4 gene plasmid by lipo- fectin regeant to package the lentiviral particles, and the viral titer was determined.The 293T cells were infected with recombinant lentivirus by different multiple of infection(MOI). The best MOI was screened with reporter gene EGFP expression.HCCLM3 cells were infected by recombinant lentivirus at the best MOI.The transfection efficiency was assessed under fluorescent microscope and NK4 protein expression levels in transfected cells were detected by Western blot method. Results The recombinant lentiviral vector of NK4 gene and EGFP gene (pLen-ti6.3-NK4-IRES-EGFP) were successfully constructed.After being packaged, purified and concentrated, the virus reached a titer of 1.08X108 TU/mL.The best MOI was 7.There was significantly expression of NK4 protein in the transfected cells, but no expression in nontranfection cells. Conclusion The NK4 recombinant lentiviral vector had been successfully constructed and effectively transfected HCCLM3 cells/The NK4 protein was highly expressed in transfected HCCLM3 cells.