The Physico-chemical Property And Functional Investigation Of The C-terminal E Peptide Of Mechano Growth Factor

Mechano growth factor (MGF) is one of alternative splicing variants of Insulin-like growth factor. MGF is comprised of 110 amino acid residues. MGF, as a growth factor and a repair factor, is strongly expressed in order to promoting local muscle regeneration and restoration.There are two isoforms of IGF-I, known as MGF and IGF-I Ea. They contain a E domain besides the B,C,A,D domains of mature IGF-I.The difference of amino acid sequence between MGF and IGF-I Ea is the C-terminal 24 amino acids of E domain. Previous research indicated that IGF-I and IGF-I Ea could promote cell proliferation and differentiation, but MGF inhibited terminal differentiation. The main purpose of this study was to investigate the characterization and function of MGF and MGF E domain.This thesis consists of three parts:Part one:The expression and purification of MGF and MGF E peptides. MGF and MGFc40 gene were cloned into plasmid pRSETc and MGFc24 was cloned into plasmid pGEX-6P-1. Then these recombinant expression plasmids were transformed with E. coli BL21(DE3). The fusion proteins were expressed at 37℃induced by IPTG. His-MGF and His-MGFc40 were solubilization and purified by His60 Ni SuperflowTM Resin. GST-MGFc24 was purified by Glutathione sepharose 4B.Part two:The study about the self-assembly characterization of MGF. In the process of the purification of MGF and MGF E peptides, we found that they were prone to aggregate. Here, we investigated the interaction of individual domain of MGF by in vitro pull down technique. The results showed that MGF and MGF E peptides could interact by themselves, while the N-terminal of MGF could interact with MGFc24. The N-terminal of MGF did not interact by itself. These results suggested the aggregation of MGF may be due to the interaction of MGF E peptides itself and the N-terminal with the C-terminal 24 amino acid residues in MGF. Part three:The study about the effect of MGF and MGF E peptide on C2C12 myoblasts. First, the C2C12 cells were treated with different concentration of His-MGF,His-MGFc40 and MGFc24. The activity of proliferation and differentiation was analyzed by CCK-8 and Creatine Kinase(CK) method, respectively. The results showed that His-MGF,His-MGFc40 and MGFc24 could promote the C2C12 cells proliferation and inhibited differentiation. Secondly, we detected the influence of MGF and MGF E peptide on Ca2+ concentration and ERK1/2 signal pathway in C2C12 cells. We found that the Ca2+ concentration was markedly increased and the ERK1/2 signal pathway was activated significantly when the C2C12 cells were treated with MGF and MGF E peptide. Thus, MGF and MGF E peptide could regulate the proliferation and differentiation of C2C12 cells though Ca2+ signal pathway and ERK1/2 signal pathway.