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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胞外段cDNA与pProEXHT载体连接 ,重组体引入大肠杆菌并在异丙基 β D 硫代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CD3 4+ 细胞的增殖情况。结果 rhFL的表达量约占菌体蛋白总量的 15 % ,经亲和纯化纯度达 90 %以上 ;rhFL +G CSF +Epo刺激CD3 4+ 细胞增殖约 40 0倍。结论 获得了rhFL在大肠杆菌中的高效表达 ,并初步建立了产物纯化途径 ,纯化后的rhFL具有较强的刺激造血干 /祖细胞增殖的能力
Objective To establish a prokaryotic expression system for recombinant human Flt3 ligand (rhFL) and purification pathways for target proteins, to provide large-scale access to rhFL products, and to promote the application of new technologies such as in vitro expansion and transplantation of stem cells in clinical applications. Methods The FL extracellular segment cDNA was ligated with pProEXHT vector. The recombinant was introduced into E.coli and induced by isopropyl β D thiogalactoside. The inclusion body was extracted and treated with metal ions after denaturation and renaturation. The purified product was purified by chromatography and the proliferation of CD3 4+ cells stimulated with rhFL was observed. Results The expression level of rhFL accounted for about 15% of the total bacterial protein, and the affinity purified purity reached more than 90%. The proliferation of CD34+ cells stimulated by rhFL + G CSF + Epo was about 40 times. Conclusion The high expression of rhFL in E. coli was obtained and the product purification pathway was preliminarily established. Purified rhFL has a strong ability to stimulate the proliferation of hematopoietic stem/progenitor cells.