FGF21基因优化及其生物活性研究

成纤维细胞生长因子21(FGF21)是FGF家族的一员,它具有独立、安全以及有效地调节生物体内血糖水平的能力。为了提高FGF21蛋白的活性,将人FGF21(hFGF21)与鼠FGF21(mFGF21)的功能区互换,构建了一种新的FGF21基因(命名为hmFGF21)。通过基因重组的方法构建SUMO-hmFGF21表达载体后,转化大肠杆菌Rosetta中诱导表达。对融合蛋白进行纯化和酶切,获得的hmFGF21突变体纯度在95%以上,并在细胞水平和动物水平上检测了突变体的生物学活性。结果表明,与野生型hFGF21相比,相同菌量hmFGF21可溶性蛋白表达量提高了约2倍。人肝细胞模型HepG2的糖吸收实验显示,hmFGF21的葡萄糖吸收显著优于野生型hFGF21。2型糖尿病动物速效降糖实验和长效降糖实验表明,hmFGF21的降糖效果显著优于野生型hFGF21。这些结果说明,野生型hFGF21经优化改造后显著提高了其生物学活性。

Optimization and characterization of a novel FGF21 mutant

Fibroblast growth factor 21(FGF21) is a member of FGF family.It has been demonstrated that FGF21 is an independent,safe and effective regulator of blood glucose levels in vivo.In order to improve the activity of FGF21,we exchanged the β10 β12 domain of the human FGF21 with that of the mouse FGF21 to construct a novel FGF21 gene(named hmFGF21),and then subcloned hmFGF21 gene into the SUMO expression vector to create pSUMO-hmFGF21 and transformed it into E.coli Rosetta for expression of the fusion protein SUMO-hmFGF21.Both in vitro and in vivo glucose regulation activity of hmFGF21 was evaluated.The SDS-PAGE result showed that compared with wild-type hFGF21,the soluble expression of hmFGF21 increased about 2-fold.HmFGF21 was more potent in stimulation of glucose uptake in HepG2 cells in vitro.The results of anti-diabetic effect on db/db mice demonstrated that hmFGF21 had better efficacy on controlling the blood glucose of the db/db diabetic animals than wild-type hFGF21.These results suggest that the biological properties of FGF21 are significantly improved by optimization.