重组表达人IDO基因慢病毒载体的构建及鉴定

目的:构建重组表达人吲哚胺2，3-过氧化酶（IDO）基因的慢病毒载体。方法:设计相应引物，从含有人IDO基因的cDNA文库中，利用聚合酶链反应（PCR）方法钓取人IDO基因的全长编码区片段。将目的基因与经酶切线性化的慢病毒载体pGC-FU进行定向的连接，将产物转化细菌感受态细胞。对长出的克隆进行菌落PCR鉴定，并对阳性的克隆进行测序及比对分析。重组慢病毒及辅助包装质粒共转染293T细胞，荧光显微镜下观察绿色荧光蛋白（GFP）的表达情况；采用Wester blot 检测IDO-GFP融合蛋白的表达情况；实时荧光定量PCR检测慢病毒浓缩液的滴度。结果:成功获取人IDO基因编码区序列，人IDO基因慢病毒转染质粒连接正确；293T细胞中产生慢病毒颗粒；IDO基因在细胞内稳定表达；人IDO基因重组慢病毒载体的滴度为2×108 TU/ml。结论:成功构建并包装出高滴度的人IDO基因重组慢病毒表达载体，为下一步转染目的细胞奠定了实验基础。

Construction and identification of the recombination Lentiviral vector with human IDO gene

Objective：To construct the recombination lentiviral expressing vector with human IDO gene.Methods：The full-length coding region fragment of IDO was amplified by primer designed by the cDNA library including IDO gene using polymerase chain reaction（PCR）.The target gene was directly inserted into the pGC-FU lentiviral vector,which were transformed into the Bacterium coli DH5α cells.The positive clone was identified by PCR and sequencing.The recombinant lentiviral vector and packaging plasmids phelper 1.0 and pHelper 2.0 were cotransfected into 293T cells.The expression of green fluorescent protein（GFP） was observed to evaluate gene delivery efficiency by fluorescence microscope.The levels of fusion protein IDO-GFP expression were detected by Western Blot,and the titers of lentiviral were determined by Real-time PCR.Results：The encoding sequences of IDO were successfully obtained by the analysis of PCR,the recombination lentiviral vector with human IDO gene was successfully constructed,and transfected into 293T cells to express IDO.The titer of lentivirus was 2 × 108 TU/ml.Conclusions：The recombinant lentiviral vector with high titer is successfully constructed,which can provide the basis of the future experiment.