Construction, Expression And Purification Of The Long-term Fusion Protein GGH And Its Preliminary Pharmacodynamic And Pharmacokinetics

Glucagon-like peptide-1 (GLP-1) is a 30-residue peptide hormone secreted by intestinal L-cells in response to nutrient ingestion. GLP-1 plays an important role in stimulating insulin secretion in a glucose induced manner, regulation of feeding, inhibiting glucagon secretion , increasingβcell proliferation, inhibitingβcell apoptosis and without low blood glucose dangerous with insulin and sulfonylureas hypoglycemic drugs. GLP-1 hypoglycemic unique mechanism is unparalleled to the existing anti-diabetes drugs. While, GLP-1 could easily degradated by DPPâ…£in vitro, the progress to make GLP-1 as a drug was restricted. This study is mainly focused on long-term nature of GLP-1. The main conclusions are as follows:PCR technology was employed to amplify the GLP-1and GLP-1_A2G, and then the express strains BL21 (pGEX-4T-1-GLP-1) and BL21 (pGEX-4T-1-GLP-1_A2G) were constructed. The SDS-PAGE analysis showed the molecular weight of fusion protein GST- GLP-1 and GST- GLP-1_A2G was about 29.0KDa. The target protein of GLP-1 and GLP-1_A2G was obtained by GST affinity chromatography and Superdex G75 after digested with thrombin. The protein of GLP-1 and GLP-1_A2G show a good biological activity.Bioinformatics softwares were used to calculated the rational conformations of GLP-1_A2G mutant and its concatenate, we also completed the two docking with the GLP-1 receptor molecule. The main binding amino acids were V30, L32, V36, Y69 and P90, most of which are aliphatic and heterocyclic amino acids, suggesting that the interaction between the four ligands and nGLP-1R is hydrophobic interaction. According calculating, there is no significated of acceptor binding energy between GLP-1 and GLP-1_A2G, while the acceptor binding energy of (GLP-1_A2G)₂ is larger than GLP-1 and GLP-1_A2G, indicating that (GLP-1_A2G)₂ is more fit for expression with albumin.Overlap PCR technology was employed to amplify the fusion gene GGH, the construction of recombinant expression plasmid pPIC9K-GGH was electrotransformated to Pichia expression system. The high expression strain KM71(pPIC9K-GGH) was selected under G418 resistance, which production was 162 mg/L under the inducing with methanol. Using western-blot technology to identify the fusion protein is a GLP-1 and the HSA hybrid elements.The fermentation process of high expression strain has been optimized, and the best growth condition of KM71(pPIC9K-GGH) has been determined: temperature 30℃, pH 6.0, liquid volume 50-60 mL in 500 mL flask,the initial concentration of glycerol 4.0%, the concentration of peptone 2.0%. The optimized expression condition consisted temperature 30℃, pH 6.0, liquid volume 50-60 mL in 500 mL flask, the methanol induced concentration 2.0 %. On the optimal condition, the highest cell concentration was 21.0 g/L, the largest specific growth rate reached 1.70 g/L·h, the highest production of target protein was up to 245 mg/L, and the best specific production formation rate was 3.38 mg/L·h. The result has been proved to be stable after five batches of fermentation, and the error of the five batches is no more than 5%.The major steps of fusion protein separation and purification have been determined, first centrifuge at 10000 r/min 10 min, and then use biomax membrane (10 kDa mochular weight) concentration 20 times, to decolored with resin DA101 and use Q FF to ion exchange, gel chromatography was used by Sephacryl S-200. The recovery rate of purification in entire process is 50.1%. The purity detection through SDS-PAGE and HPLC was upper than 95%, this can be in line with pharmacokinetics request.The fusion protein GGH can stimulate the growth of islet cell in vitro. When the concentration of GGH is 45 nmoL, the growth rate is 35.4%. The result was similar as GLP-1. In the glucose tolerance test, GGH fusion protein in mice can better control the blood sugar levels. After 72 h administration, biological activity of high-dose group still can be detected. However, there was no biological activity after 4 h administration GLP-1.that the, indirectly reflects 300 hours in was 16.0 percent, that part of the drug through fecal excretion.Pharmacokinetics research showed that the maximum plasma concentration reached the highest after 8 h administration the fusion protein GGH by abdominal subcutaneous, the volume of distribution was 53.4 mg/L·h, the half-life of absorbtion in vivo was 26.6 h, the elimination half-life in vivo was about 57.8 h. Subcutaneous injection of the fusion protein GGH in mice, the main organs were all reached peak in 6 h, There were higher accumulations of radioactive by the unit weight in mice stomach, kidney, lung and muscle, especially it was highest in stomach, intestine, pancreas, liver, fat and genital were followed by. What'more, there had the least distribution in heart and brain.The average cumulative fecal excretion rate in urine after 300 h was 80.1%. It is suggested that kidney was the main way to eliminate isotope, and also reflected that the drug (including metabolites) mainly egested through kidney. The average cumulative fecal excretion rate in manure after 300 h was 16.0%. It is suggested that this drug can egest by manure.