Study On Insulin-like Growth Factor-1 And Insulin-like Growth Factor Binding Protein-1

Insulin-like growth factor -1 (IGF-1) and Insulin-like growth factor binding protein-1 (IGFBP 1) are important functional proteins in human body. The interaction between IGF-1 and IGFBP—1 regulates the content and distribution of free IGF-1. It also affects growth and development, tumor occurrence and carbohydrate metabolism. However the mechanism of interaction remains unclear. IGF-1 has the insulin-like function, but the application is limited to the treatment of diabetes because IGF-1 has many bioactivi ties. A series of studies on IGF-1, IGFBP-1 and mutants of IGF-1 were performed by means of DNA recombination in vitro, yeast three-hybrid, cell cultrue, CAM. 1. Cloning and expression of IGF-1 and its mutantsThe human IGF-1 cDNA was isolated by PCR from fetal hepatic cDNA 1 ibrary and confirmed by sequence analysis. Two mutants of IGF-1, IGF97 and IGF191, were designed and constructed by bioinformatics and DNA recombination. The two mutants of IGF-l were expressed as a GST fusion protein in E. coli. The two mutants were successfully cleaved by thrombin and purified with affinity chromatography. IGF-l and two mutants were also expressed by the plasmid pcDNA3. 1+ in COS-7 cells. In order to study the biological activity of the mutants, the MTT method was used to determine the changes of proliferation of ECV304 caused by the IGF-1 and mutants. The function to stimulate cell proliferation by IGF191 was significantly decreased in comparison with IGF-1 and IGF97. The CAM technique was used to investigate the effect of IGF-1 and mutants of IGF-1 on the vascularization. The results indicated that IGF-1 and IGF97 couldpromote the CAM angiogenesis. The IGF191 cannot affect the angiogenesis. 2. Study on proteins interacted with IGF-1 and IGFBP-1 by the yeast three-hybrid systemThe method of yeast three-hybrid technique was established and firstly used to study the proteins interacted with IGF-1 and IGFBP-I. The ability of transcriptional activity of IGFBP-1 was found. The relationship between the structure and function of IGFBP-1 was analysed by bioinformatics. A mutant was designed by deleting 12 amino acid of the carboxyl end oF IGFBP-1. The mutant cDNA was obtained by PCR, characterized by DNA sequencing, and inserted into BD plasmid. The transcriptional ability of IGFBP-1 was successfully destructed by construct the mutant IGFBP-M2.Three cDNA encoding the proteins interacted with IGF-1 and IGFBP-1 were screened out of the fetal hepatic cDNA library by the yeast three-hybrid system. By homology comparison, these genes were identified as human ferritin, hemoglobin, LMW ubiquinone-binding protein.In brief, IGF-1 and the two mutants were expressed in prokaryotic and eukaryotic cells. The mutant IGF191 may be a significantly protein applied to diabetes treatment. The proteins interacting with IGF-1 and IGFBP-1 may be new cues for investigating new biological activity and mechanism of IGF-1 and IGFBP-1.