Preparation And Activity Analysis Of IGF-1 Based On Protein Structure Prediction

As a multi-functional homeostasis regulator in human body,IGF-1 plays an important role in the human body by promoting growth and development,anabolism and other functions.As the only drug for the treatment of primary IGF-1 deficiency syndrome,IGF-1 is in high demand in China.There are already IGF-1 protein drugs on the market abroad,but China still relies on imports,which has caused a great economic burden on patients,resulting in patients with growth retardation being unable to receive effective treatment.Therefore,the development of import substitution drugs is of great significance.In this research,structures of the IGF-1 fusion proteins were predicted by computer simulation technology firstly.Then,the IGF-1 fusion proteins docked with different IGF-1 proteases.The most suitable IGF-1fusion protein for protease was screened.The prokaryotic expression vector of IGF-1 fusion protein was constructed and identified by genetic engineering technology.Recombinant plasmid was transformed into Escherichia coli Origami B(DE3)strains and selected out by ampicillin resistance.IPTG was induced expression.The bacterial supernatant was purified by two-step affinity chromatography,gel filtration to remove salt and thrombin cutting.Finally,3T3 cell activity assay was established and activity of the prepared IGF-1 was tested.Results showed that the fusion protein was inducible expressed in the condition of E.coli Origami B(DE3),0.05M IPTG,induced at 25?for 16 h.In the enzyme buffer of 20 mM Tris-HCl+0.15 mM NaCl+10 mM CaCl₂+1 mM DTT,100?g fusion protein could be cutted by 1 U thrombin at 25?and for 16 h.The purity of IGF-1 protein was more than 90%by two-step affinity chromatograpy.The cell activity assay was established by 3T3 cell proliferation method.The cell density of detected condition was 1×10⁴/mL.The initial,ceiling and maintain FBS concentrations of culture medium were 4%,2%and 1%,respectively.The initial concentration of IGF-1 sample was 1000ng/mL.The dilution gradient was 3.Under this activity assay,the specific activity of IGF-1 prepared was 2.47×10⁵U/mg,close to the market standard.This study can provide experimental basis for industrial production of IGF-1 drugs.