重组人Flt3配体在大肠杆菌中的表达、纯化与功能鉴定

目的：建立重组人Flt3配体（rhFL)的原核高效表达系统及目标蛋白的纯化途径，为大规模获得rhFL产品，促进干细胞体外扩增，移植等新技术在临床的应用创造条件，方法：将FL细胞段cDNA与pProEXFT载体连接，重组体引入大肠菌菌并在异丙基－β－D－硫化半乳糖苷诱导下表达，提取包涵体，经变性，复性等处理，用金属离子螯合层析纯化表达产物，观察纯化所得rhFL刺激CD^ 34细胞的增殖情况，结果：rhFL的表达约占菌体蛋白总量的15％，经亲和纯化纯度达90％以上，rhFL CG-CSF＋Fpo刺激CD34^ 细胞增殖约400倍，结论：获得了rhFL在大肠肝菌中的高效表达，并初步建立了产物纯化途径，纯化后的rhFL具有产强的刺激造血干／祖细胞增殖的能力。

Expression of recombinant human Flt3 ligand in Escherichia coli and its purffication and characterization

OBJECTIVE: To establish a highly efficient expression system of recombinant human Flt3 ligand (rhFL) in E. coli and a suitable purification method of the expressed products. METHOD: Human FL encoding cDNA was introduced into pProEXHT plasmid to express a 6 x His-FL fusion protein in E. coli. The fusion protein expressed in inclusion body was isolated, solubilized and refolded, and then purified by chromatography on a metal-chelating affinity column (MCAC). Its activity was detected by stimulating the proliferation of CD(34)(+) cells. RESULT: The amount of rhFL expressed was about 15% of total bacterial proteins and the purity of rhFL was 90% after MCAC. The combination of rhFL, granulocyte colony-stimulating factor and erythropoietin could stimulate CD(34)(+) cells to a 400 fold expansion. CONCLUSION: The purified rhFL had a potent activity to stimulate hematopoietic stem cells to expanse in vitro.