过表达吲哚-2,3双加氧酶慢病毒载体的构建

Constructing a lentiviral vector overexpressing indoleamine 2,3-dioxygenase

背景:器官移植后的免疫排斥反应或免疫抑制药物严重不良反应使患者得不到有效治疗、治疗效果不佳。基于此背景下,结合最新免疫调节研究结果,试图寻找到一种有效的生物免疫抑制方法。目的:构建过表达吲哚胺2,3-双加氧酶(indoleamine 2,3-dioxygenase,IDO)慢病毒载体。方法:1将已构建成功的IDO基因插入慢病毒包装质粒GV308中,构建GV308-IDO重组慢病毒包装质粒。2用慢病毒包装辅助元件Psi、c PPT、3FLAG、Tet R、IRES、WRPE、Tet IIP、Ubiquitin Promoter、SV40origin及HIV的基本元件5’LTR和3’LTR,共培养法转染80%融合的293T细胞。结果与结论:Western blot检测经10 g/L琼脂糖凝胶电泳可见在Mr 48 000处有一目的片段,该值与IDO蛋白大小一致。RT-PCR检测可见293T细胞中有IDO基因表达。说明IDO融合基因已经成功重组于慢病毒包装质粒内。

BACKGROUND： Immune rejections after organ transplantation or serious adverse reactions due to immunosuppressive drugs show a lack of effective treatments and poor therapeutic outcomes. Therefore, we try to find an effective immune suprresion method in combination of the latest immunomodulatory achievements. OBJECTIVE： To construct a lentiviral vector overexpressing indoleamine 2,3-dioxygenase（IDO）. METHODS：（1） The IDO gene that was successfully contructed was inserted into lentiviral packaging plasmids GV308 to construct GV308-IDO lentivirus packaging plasmids.（2） The 293 T cells with 80% confluence were co-cultured with 5＇LTR and 3＇LTR, basic elements of lentiviral packaging auxiliary components, including Psi, c PPT, 3FLAG, Tet R, IRES, WRPE, Tet IIP, Ubiquitin Promoter, SV40 origin and HIV. RESULTS AND CONCLUSION： Western blot assay showed that in 10 g/L agarose gel electrophoresis, there was a target fragment at Mr 48 000. This value was consistent with the size of IDO protein. RT-PCR results showed visible IDO expression in 293 T cells. These findings suggest that IDO fusion gene has been successfully reorganized in the lentiviral packaging plasmids.