人FGF-21基因的克隆、表达及其调节脂肪细胞糖代谢的活性

Cloning, expression and glucose regulation activity of human FGF-21

成纤维细胞生长因子FGF-21是FGF家族的一个新成员,最近的研究发现其具有调节血糖的作用,有望成为治疗2型糖尿病的基因药物。文章应用RT-PCR技术,从成人肝脏中克隆人源的FGF-21成熟蛋白基因,并将其克隆到T载体上,经测序鉴定后,将其亚克隆到原核表达载体pSUMO上,转入大肠杆菌Rosetta(DE3)中。鉴定阳性克隆后,用IPTG诱导FGF-21表达,并用Ni-NTA柱进行亲和层析纯化。以3T3-L1脂肪细胞的葡萄糖吸收实验来鉴定FGF-21表达产物促进糖吸收的活性。结果表明,FGF-21成熟蛋白基因大小为546bp,测序结果与GenBank数据库中的序列一致。SDS-PAGE与Western blotting结果表明:人源FGF-21成熟蛋白大小19.4kDa,经3T3-L1脂肪细胞的葡萄糖吸收实验验证其具有促进葡萄糖吸收的生物活性,并且GLUT1是FGF-21发挥生物学作用的终末执行单位。

Fibroblast growth factor （FGF）-21 is a recently discovered glucose regulator and has potential to become therapeutics for treatment of type 2 diabetes. The aim of this study was to clone and express human FGF-21 gene and characterize its bioactivity for glucose regulation. The hFGF-21 cDNA was cloned from human liver by RT-PCR and subcloned into the pSUMO vector after sequencing confirmation. The recombinant plasmid was transformed into Escherichia coli Rosetta strain. The FGF-21 protein expression was induced by IPTG and purified by Ni-NTA agarose. The FGF-21 product was verified by Western blotting analysis with specific antibody. The bioactivity of the purified protein was examined by glucose uptake assay in 3T3-L1 adipocytes. The cloned hFGF-21 gene consisted of 546 bp, which was in agreement with the published data in GenBank. SDS-PAGE analysis showed that hFGF-21 expressed in the E. coli system was 19.4 kDa in size. The glucose uptake assay in 3T3-L1 adipocytes indicated that the purified hFGF-21 could stimulate glucose uptake in a dose-dependent manner, and glucose transporters （GLUT1） is the functional unit.