The Study Of FGF21Cloning, Expression And Promote Glucose Absorption

FGF21is a member of the fibroblast growth factor family (FGFs) which has been found recent years. FGF21and its FGF subfamily members has become the focus of glucose and lipid metabolism. It shows the protective effect on glucose and lipid toxicity and cytokine-inducedislet (3cell apoptosis, and plays an important role in control the secretion of the beta cells of the pancreasguided activities. FGF21is an effective factor of blood sugar balance regulation.FGF21promotes glucose metabolism, reduce the triphosphate content of glycerol and glucose to the body’s normal level, and shows the function of control of blood glucose, protect the number and function of pancreatic β-cell conductivity. Wolf et. Al. found that FGF21increase the activity of pancreatic β-cell conductivity and pancreatic guide beta cells through activate extracellular signal-regulated kinase1/2and Akt signaling pathways.Purpose:Expression a large number of FGF21protein with high biological activity through E. coli expression system, and successfully purified protein to get pure FGF21, study its efective of promote glucose absorption on Liver cells, muscle cells, preadipocytes and of ob/ob mice.Methods:Synthesis of FGF21DNA fragments, build PET-3C-FGF21vector, transformed the vector into E. coli. to expression the FGF21protein.Target protein was purified by three-step column chromatography to get pure protein. Cultured normal human liver cells (HL-7702), rat muscle cells (L-6), murinepreadipocytes (3T3-L1), determined cells glucose uptake with GOD-POD kit. FGF-21daily0.6μg·g-1dose subcutaneous continuous injection for7days, respectively, administration of fasting blood glucose on day0, day3, and day7,with saline control; rhFGF-21daily0.6μg·g-1dose subcutaneous continuous injection9days, testoral glucose tolerance1h after the last administration, oral administration of2mg·g-1glucose solution, test blood glucose after30min,60min,120min of given glucose solution; rhFGF-21daily2.5μg·g-1dose subcutaneous injection for seven consecutive days, detect postprandial blood glucose after administration of1h in day0, day3and day7.Results:Construction and sequencing recombinant vector correct, shows that the PET-3C-FGF21vector was successfully constructed. Through optimization expression conditions and protein detection, indicating thatprotein expression of inducible expression conditions for OD600=0.8～1.0, of IPTG concentration is1.OmM,37℃induced four hours. Most of the expression in supernatant, purification of FGF21protein by hydrophobic interaction chromatography, ion exchange chromatography, gel filtration chromatography, HPLC analysis showed high purity of the protein, Western blot analysis showed that the protein have strong immunogenicity with FGF21antibody. In vitro experiments and animal experiments show that3.9mg·L-1～100mg·L-11FGF21promote HL-7702cells in glucose absorption and a concentration-effect relationship, with the control group the difference was significant (*P<0.01); FGF21in L6muscle cells to absorb glucose,0.3mg L-1～250mg·L-1promote significant difference(*P<0.05)compared with the control group;12.6mg of L-1～100mg·L-1FGF21promote differentiation of3T3-L1cells inglucose absorption and a concentration-effect relationship, compared with the control group, significant differences (*P<0.05); ob/ob mice blood glucose test proved that rhFGF-21has a role in lowering blood glucose and improve glucose tolerance.