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成纤维细胞生长因子21(fibroblast growth factor21,FGF21)是2000年发现的一个不依赖胰岛素调节血糖的细胞因子,有望成为治疗糖尿病的候选药物.但是,野生型FGF21由于半衰期较短,在体内不稳定,从而影响其成药性.为提高FGF21的稳定性,本实验在FGF21的C端添加了2个精氨酸(arginine,Arg),命名为FGF21-2A,用Compute pI/Mw软件计算之后,等电点(isoelectric point,pI)从5.43上升到5.84,随后进行了蛋白质的表达、分离纯化、体内稳定性及糖代谢调节作用的研究.诱导表达后菌液的SDS-PAGE图经BandScan5.0分析后显示FGF21-2A的表达量相对于野生型FGF21提高了10.6%.家兔体内半衰期检测实验结果显示FGF21-2A的半衰期显著延长.GOD-POD法检测HepG2肝癌细胞葡萄糖吸收实验、糖尿病小鼠降血糖实验和肝糖原检测实验的结果证明,FGF21-2A的降糖效果得到了增强,并且持续时间相对于野生型FGF21显著延长.Real-time PCR结果发现,长期注射FGF21-2A显著提高了糖尿病小鼠肝脏内葡萄糖转运蛋白(GLUT)1和葡萄糖激酶(GK)mRNA的表达量,降低了葡萄糖-6-磷酸酶(G6P)的mRNA表达量,表明FGF21-2A调节糖代谢的机制与野生型FGF21一致.综上所述,精氨酸修饰的FGF21其蛋白质稳定性提高,进而增加了对血糖的调控效果,有望成为新型糖尿病药物.
FGF21, a non-insulin-dependent regulator of blood glucose, was found to be a potential drug candidate for diabetes in 2000. However, wild-type FGF21 is unstable in vivo due to its short half-life , Thus affecting its drug-induced.In order to improve the stability of FGF21, we added two Arginine (Arg) at the C-terminus of FGF21 and named FGF21-2A, calculated with Compute pI / Mw software, etc. The isoelectric point (pI) increased from 5.43 to 5.84, and then the protein expression, isolation and purification, in vivo stability and the regulation of glucose metabolism were studied.After induced expression, SDS-PAGE of the bacterial liquid was analyzed by BandScan 5.0 The results showed that the half-life of FGF21-2A was significantly prolonged.GOD-POD method was used to detect the glucose uptake in HepG2 hepatocarcinoma cells and the level of FGF21-2A expression was decreased by 10.6% The results of the blood glucose test and the liver glycogen test showed that the hypoglycemic effect of FGF21-2A was enhanced and the duration was significantly longer than that of wild-type FGF21. Long-term injection of FGF21-2A significantly increased the mRNA expression of glucose transporter (GLUT) 1 and glucokinase (GK) in the liver of diabetic mice and decreased the mRNA expression of glucose-6-phosphatase (G6P), indicating that FGF21 -2A regulation of glucose metabolism is consistent with that of wild-type FGF21 In conclusion, the stability of arginine-modified FGF21 protein is improved, which in turn enhances the regulation effect on blood glucose and is expected to become a new type of diabetes drug.