Optimization Of Prokaryotic Expression Conditions Of Human GDF-15 And Preliminary Study On Hypoglycemic And Lipid-lowering Effects

Objective:To construct the pET28a-hGDF15 prokaryotic expression vector;To induce the expression of hGDF-15 protein and to optimize some expression condition in the early stage of the laboratory based on the optimized again.The protein was injected to diabetes mice to observe the effect of lowering sugar and lipid and to ?lay the foundation forseeking the target of hGDF-15.Methods: To construct the Recombinant pET28a-hGDF15 Prokaryotic Expression Vector by using Molecular Biology Related Techniques;Induced to induce expression of the recombinant plasmid;To optimized the expression conditions on the basis of the previous work in the laboratory and to analyzed the optimal expression conditions in supernatant and sediment.The protein samples were identified by Western blot with DAB chromogenic method,and the expressed product was purified by nickel column.The target proteins were eluted with imidazole at concentrations of 20,100,and 300 mmol/L;The purchased C57BL/6 male mice were randomly grouped.Experimental group was given high-fat diet(high-fat feed was purchased from Beijing Keao team),and the Control group was given basic feed.After 4 weeks,STZ was injected into experimentally qualified mice(100mg).One week later,the successful animal models of insulin resistance were selected which the fasting blood glucose values greater than 7.8 mmol/L.Injected the the mice hGDF-15 proteinwere treated by subcutaneous injection of hGDF-15 protein and observed the changes of the corresponding indicators after medication.Result: Constructed the pET28a-hGDF15 prokaryotic expression vector was successfully constructed;hGDF-15 protein was successfully induced;The results showed that when the concentration of IPTG was 0.75 mmol/L,the temperature was 37°C and the time was 8 h,the content of inclusion body protein was the highest.The target protein was confirmed by Western blot and the target protein was successfully eluted with 300 mmol/L imidazole.The high-fat insulin-resistant C57BL/6 mouse mode lwas successfully established and proved that hGDF-15 has some effects on hypoglycemic and lipid-lowering.Conclusion: We successfully constructed the expression vector ofpET28a-hGDF15 prokaryotic,induced the expression of protein,optimized optimal conditions for induction,and purified the protein.By using the proten to treat ?diabetes mice.we found that the mice's body weight,blood glucose,blood lipids,glucose tolerance and other indicators have improved,providing experimental basis for the study of the function of the protein.