Flt3配体胞外域的原核表达纯化及对脐血CD34+细胞的扩增作用*

背景：Fms 样酪氨酸激酶3配体(Flt3配体)是一种重要的生长因子，通过激活特定的酪氨酸激酶受体，调控造血细胞的生长、生存和/或分化，具有促进造血干细胞体外扩增的应用潜力。目的：构建pET32a(+)-hFLext原核表达载体，表达、纯化hFLext蛋白，观察其对脐血CD34+细胞的扩增作用。方法：克隆hFLext，构建pET32a(+)-hFLext重组表达载体。转化大肠杆菌 BL21，IPTG诱导蛋白表达，镍珠亲合层析纯化蛋白。磁珠分选脐血CD34+细胞，单独加入hFLext或联合干细胞因子、血小板生成素孵育1周，观察体外扩增作用。结果与结论：成功克隆hFLext，并构建了pET32a(+)-hFLext重组表达载体。在大肠杆菌BL21成功表达Trx-hFLext融合蛋白，经8 mol/L尿素变性包涵体蛋白，逐步透析复性，镍珠亲合层析纯化蛋白，成功获得高纯度的Trx-hFLext融合蛋白。Trx-hFLext融合蛋白不仅具有维持及轻度刺激CD34+细胞体外扩增的作用，并且与干细胞因子及血小板生成素具有协同作用，为造血干/祖细胞体外扩增研究奠定了基础。

Prokaryotic expression and purification in Flt3 ligand extracellular domain and effects on cord blood CD34+ cells amplification

BACKGROUND: Fms-like tyrosine kinase 3 ligand (Flt3 ligand, FL) is a cytokine that affects the growth, survival and/or differentiation of hematopoietic cells through the activation of specific tyrosine kinase receptors, and it is potentially useful for in vitro hematopoietic stem cells (HSCs) amplification.OBJECTIVE: To construct the recombinant prokaryotic expression vector pET32a(+)-hFLext, and to express and purify hFLext protein and investigate its effect on expansion of cord blood CD34+ cells. METHODS: The hFLext gene was cloned and the recombinant expression vector pET32a-hFLext was constructed and transformed into E. coli BL21. The hFLext protein was expressed under IPTG induction, purified by affinity chromatography with Ni beads. The cord blood CD34+ cells were isolated using CD34 microbead kit and incubated with hFLext alone or in combination with SCF and TPO. The effect of hFLext on expansion of CD34+ cells was examined after 1-week incubation. RESULTS AND CONCLUSIONS: The recombinant expression vector pET32a-hFLext was successfully constructed and the hFLext was successfully expressed as a Trx fusion protein (Trx-hFLext) in E. coli BL21. The Trx-hFLextwith high purity was successfully obtained after degenerating proteins in inclusion bodies with 8M urea, renaturing by gradual dialysis, purifying by affinity chromatography with Ni beads. The purified hFLext can not only effectively maintain and slightly stimulate the proliferation of cord blood CD34+ cells, but also expand profoundly in combination with SCF and TPO, thus paving the way for its application in expanding human hematopoietic stem/progenitor cells in vitro further.