The Construction Of A Mutant IGF-1 And Evaluation Of Its Biological Function

Insulin-Like Growth Factor-1(IGF-1)is a single-chain polypeptide consised of 70 amino acid residues.It is an important growth stimulating factor that was mainly synthesized and secreted by liver cells and played an important physiological role in a variety of cells.IGF-1 acted on target organs to regulated cells biological behavior through autocrine?paracrine and endocrine processes,mediated by insulin-like growth factor receptor 1(IGF-1R)and insulin-like growth factor binding protein(IGFBP).A large number of studies have shown that IGF-1 and IGF-1R showed high levels of expression in a variety of malignant tumors such as breast cancer,uterine cancer,prostate cancer,and non-small cell lung cancer.Once IGF-1 binding to IGF-1R,the phosphorylation of specific tyrosine residues of IGF-1R was induced,then binded to the aptamer molecule Shc and insulin receptor substrate-1,which leaded to the activation of phosphoinositide 3-kinase and mitogen-activated protein kinase cascade signaling pathway,to promoting the proliferation and migration of tumor cells.Therefore,IGF-1 is one of the targets for cancer treatment.In recent years,a number of targeted therapeutic drugs that targeted IGF-IR have been developed,including siRNAs ?monoclonal antibodies and kinase inhibitors to inhibit the enzymatic activity of receptors,which provided a thought that an IGF-1 mutant can be designed as a competitive inhibitor of IGF-1 to bind to IGF-1R competitively,which can regulate certain biological behaviors of tumor cells without affecting other biological behaviors.In this study,we analyzed and collated the published literature and analyzed the important sites that may affect the binding of IGF-1,receptor IGF-1R,integrin ?v?3 and binding protein IGFBP.These sites were mutated to designed a mutant IGF-1 that was used to study the effect of the mutant IGF-1 about the proliferation and migration of human hepatoma cell Hep.G2.In addition,we studied the expression of the recombinant protein and optimized its process.The research could provide the experimental basis for the application development of the mutant IGF-1.The main research contents and conclusions of this paper are as follows:(1)IGF-1 gene was mutated by point mutation PCR and overlap PCR.Wild-type IGF-1 and mutant IGF-1 eukaryotic expression plasmids were successfully constructed.(2)The recombinant plasmid was transfected into human hepatocellular carcinoma Hep.G2 cells by non-lipid-mediated plasmid transfection.The expression of fluorescent protein of the vector was observd and the expression of fusion protein was detected by ELISA assay,which confirmed that the recombinant plasmid was transfected and the target protein was expressed successfully.The Hep.G2 cell proliferation was detected by MTT assay and the Hep.G2 cell cycle was detected by flow cytometry,then the Hep.G2 cell cell migration was detected by Wound Healing assay,in addition,the expressions of MMP9 and VEGF in Hep.G2 cell were also detected.The results displayed that the recombinant plasmid was transfected into Hep.G2 cells and the recombinant protein was expressed successfully.The mutant IGF1 did not promote the Hep.G2 cell proliferation and there was no obvious correlation with the change of cell cycle.It has a significant inhibitory effect on Hep.G2 cell migration,and the expression of VEGF in Hep.G2 cell was down-regulated.(3)The recombinant plasmid pET-28a-IGF-1 was transformed into the expression vector E.coli BL21(DE3).The target protein was induced by IPTG and the induction conditions were optimized.The induction temperature was 37°C and the induction time was 6 hours,the IPTG concentration was 0.6 mM and the target protein was purified by affinity chromatography.