人FLT3配体的克隆、表达与纯化

Gene cloning, expression and purification of human FLT3 ligand

目的 克隆人 FL T3配体 (Flt3L igand,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 FL T3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 p RSET- B;重组质粒 p RSET- B- FL 转化大肠杆菌 BL2 1(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 4 6 2bp的 FL基因 ,测序正确 ;经 Eco R 和 H ind 酶切鉴定证实 ,FL 成功地克隆到表达载体 p RSET- B;构建的表达载体p RSET- B- FL在大肠杆菌中表达出 Mr 2 4 0 0 0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 .

AIM To clone human FLT3 ligand gene, express FL protein in E.coli and purify it preliminarily. METHODS A cDNA encoding soluble FL was cloned through RT nested PCR from the total RNA extracted from human peripheral blood mononuclear cells and identified by analyzing the nucleotide 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 FLT3 ligand 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. SDS PAGE and Western blot were employed to identify the expression of human FLT3 ligand. RESULTS FL gene with a length of 462 bp 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 Eco RⅠ and Hin dⅢ enzyme. SDS PAGE showed that a M r 24 000 fused protein was expressed in E.coli . The purity of the fused protein obtained by ion exchange method was 90%. 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 expressed in E.coli . The fused protein was purified preliminarily. The study will provide necessary conditions for obtaining rhFL protein on a large scale.