摘要
目的构建携带鼠Foxp3基因的自身失活型慢病毒载体,并研究其在CD4+CD25-T细胞中的表达。方法构建含鼠Foxp3基因及内部核糖体进入位点序列(IRES)和绿色荧光蛋白基因(GFP)的双顺反子慢病毒载体。采用脂染法将慢病毒载体三质粒转染293T细胞,经共培养法感染免疫磁珠分离的小鼠CD4+CD25-T细胞,流式细胞术(FCM)检测基因转导效率,通过RT-PCR及Western Blotting法分别从mRNA和蛋白水平检测Foxp3的表达。结果成功构建了慢病毒表达质粒pXZ208-IRES-GFP/pXZ208-Foxp3-IRES-GFP,包装的重组慢病毒滴度达106IU/mL。以pXZ208-IRES-GFP为阴性对照组,共培养法感染CD4+CD25-T细胞,实验组细胞基因转导效率达55.95%,并且存在Foxp3 mRNA和蛋白水平的高表达。结论成功构建了携带鼠Foxp3基因的自身失活型慢病毒载体;该系统可有效的介导Foxp3基因在CD4+CD25-T细胞中表达。
Objective To construct the self-inactivating lentiviral vector containing murine Foxp3 gene and detect its expression in the CD4^＋ CD25^- T cells. Methods The bicistronic lentiviral transfer plasmid containing Foxp3 gene and internal ribosomal entry site-green fluorescent protein gene （IRES-GFP） was constructed. Human embryonic kidney 293T cells were co-transfected by lentiviral packing system, which including the packaging plasmid ΔNRF, the transfer plasmid and the envelope plasmid VSVG using liposome. Then manifested the efficiency of gene transduction with Flow Cytometry （FCM） and the expressions of Foxp3 mRNA and protein were assayed by RT-PCR and Western blotting. Results The lentiviral transfer plasmid pXZ208-Foxp3-IRES-GFP was constructed, the virus titres were above 106 IU/mL in the supernatant. After coculture with thus cells, the CD4^＋ CD25^-T cells in experimental group expressed significantly higher amounts of Foxp3 mRNA and protein than control group, and the efficiency of gene transduction was about 55. 95%. Conclusions The self-inactivating lentiviral vector containing murine Foxp3 gene are successfully constructed. This gene delivery system can transduce Foxp3 gene into CD4^＋ CD25^- T cells with highly efficient expression.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2009年第2期199-202,共4页
Journal of Sichuan University(Medical Sciences)
基金
国家自然科学基金(批准号30770915)
江苏省自然科学基金创新人才项目(BK2007504)
江苏省普通高校研究生科研创新计划(CX07S-039Z)资助
