Cloning, Optimization Of Expression Conditions And Pharmacological Activity Of HGDF-15

Objective: To clone the mature peptide gene of human growth differentiation factor-15 and construct prokaryotic expression vector, induce the expression of the hGDF-15 gene and optimize expression conditions. The results of expression products were analyzed by Western blotting, the hGDF-15 protein was purified by affinity of Ni- Agarose and His tag. The animal model induced by high fat diet and STZ to insulin resistance. To observe the effect on the successing animal model of injection of hGDF-15 protein, and lay the foundation for research on pharmacological activity and biological function of hGDF-15.Method: The hGDF-15 mature peptide gene was cloned by using PCR technology and correctly inserted into the vector pET-28 a to construct pET-28a-hGDF-15 prokaryotic expression vector. Plasmid containing pET-28a-hGDF-15 gene was transformed into E.coli Rosetta(DE3) competent cell to obtain recombinant strain.SDS-PAGE analysis of the induced expression of hGDF-15 gene under different temperatures(16,25,37℃),different concentrations of IPTG(0.1,0.25,0.1,0.25,1mmol/L) and different duration(12,24,36,48h).Using Gel Quant Express software to determine the content of centrifugal supernates and precipitates in the crushed bacteria. The optimal expression conditions of supernates and precipitates were analyzed by orthogonal experiment.Using Western blot tecnology to identify the induced protein samples by DAB coloration method. The expression products of hGDF-15 gene were purified by affinity of Ni-Agarose with different concentrations of GuNTA(20,40,60,100,500 mmol/L imidazole) and freezed drying.The experiment C57BL/6 mouses were fed with high fat diet(80% basic forage, 10% lard, 10% yolk), and the control were fed with basic forage. Insulin Resistance Animal Model were established by injecting with STZ(100 mg/kg) to the qualified weight experiment mouses. The hGDF-15 protein used for treatment by Subcutaneous injection, and the effect of it was observed by the change of blood glucose after treatment.Result: The hGDF-15 mature peptide gene(339 bp) was successfully cloned into pET28 a prokaryotic expression vector. According to the orthogonal experiment to optimize the optimal expression conditions,the supernatant was expressed by 0.25 mmol/L IPTG for 24 h at 16 and ℃the precipitate was expressed by 1 mmol/L IPTG for 36 h at 37℃. The hGDF-15 proteins were confirmed by Western blotting. The hGDF-15 were purified by affinity of GuNTA(100 mmol/L). The recovery rate of 29.8% calculated after purification. Insulin resistance was established in C57BL/6 mouse. We show that hGDF-15 has a certain role in treatment of diabetes and insulin resistance.Conclusion: The hGDF-15 mature peptide gene(339 bp) was successfully cloned, the hGDF-15 proteins were expressed and the optimal expression conditions were optimized. The hGDF-15 protein was obtained, and has proved vital in insulin resistance.