人FLT3配体的克隆、表达与纯化.docVIP

人FLT3配体的克隆、表达与纯化 作者：吴成利　师建国　颜真　韩苇　石继红　张英起 【关键词】 基因表达 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. 　　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），and then FLT3ligand was expressed under the inducement of IPTG.The fused protein was purified by ion exchange methods of SP-Sepharose Fast Flow and Q-Sepharose Fast Flow.SDSand Western-blot were employed to identify the ex-pression of human FLT3ligand.RESULTS FL gene with a length of462bp was isolated from human peripheral blood mononuclear cells.Sequence analysis revealed the sequence of FL gene was consistent with relevant reports.FL gene was cloned into the expressing vector pRSET-B identified by enzyme digestion of EcoRⅠand HindⅢenzyme.SDSshowed that a Mr24000fused protein was expressed in E.coli.The purity of the fused protein obtained by ion exchange method was90%.Wenstern-blot showed the fused protein could be recognized by the anti-FL antibody.CONCLUSION The FL gene was obtained by RT-PCR amplification.The expressing vector of FL was successfully constructed and ex-pressed in E.coli.The fused p