关键词: 配体;克隆，分子;基因表达;蛋白纯化

    摘 要:目的 克隆人FLT3配体(Flt3Ligand，FL)基因，并在大肠杆菌中对FL进行表达、纯化. 方法 分离人外周血单个核细胞，提取总RNA，采用RT-巢式PCR扩增人FLT3配体胞外段基因，以双脱氧终止法进行DNA序列分析;将测序正确的目的基因克隆入大肠杆菌融合表达载体pRSET-B;重组质粒pRSET-B-FL转化大肠杆菌BL21(DE3)，用IPTG进行诱导后，收集细菌，菌体裂解后，利用常压离子交换色谱的方法对表达蛋白进行纯化，并进行SDS-PAGE及Western-blot检测. 结果 从人外周血单个核细胞中克隆得到长462bp的FL基因，测序正确;经EcoRⅠ和HindⅢ酶切鉴定证实，FL成功地克隆到表达载体pRSET-B;构建的表达载体pRSET-B-FL在大肠杆菌中表达出Mr24000的融合蛋白，经常压离子交换色谱化后的融合蛋白的纯度达到90%，该融合蛋白具有FL抗原特性. 结论 成功地克隆并表达了FL基因，并对融合蛋白进行了初步纯化，为大规模获得FL创造了条件.

    Gene cloning，expression and purification of human FLT3ligand

    WU Cheng-Li SHI Jian-Guo YAN Zhen HAN Wei SHI Ji-Hong ZHANG Ying-Qi

    1 Department of Pathology，Faculty of Preclinical Medicine，

    2 Biotechnology Center，Science Research Administration，Fourth Military Medical Universi-ty，Xi'an710033，China

    Keywords:ligands;cloning，molecular;gene expression;protein purification

    Abstract:AIM To clone human FLT3ligand gene，express FL protein in E.coli and purify it preliminarily.METHODS A cDNA encoding soluble FL was cloned through RT-nest-ed PCR from the total RNA extracted from human peripheral blood mononuclear cells and identified by analyzing the nu-cleotide sequences.The human FL gene was subcloned into the expressing vector pRSET-B.The recombinant plasmid pRSET-B-FL was transformed to BL21(DE3) ......