The Reorganization Epoab-ngf Mimetic Peptide Expression In Escherichia Coli, Purification And Biological Activity Of A Preliminary Study

Erythropoietin (Epo) was originally identified as the principal regulator of erythroid progenitor cells. It was newly found that Epo exerted cytoprotive and nutritional effects in neural system and blood vascular systerm which does not depend on erythopoiesis. Moreover, this nerval protective and nutritional action has been positioned in a polypeptide sequence composed of seventeen amino acids named EpoAB peptide (AEHCSLNENITVPDTKV).Nerve grow factor (NGF) can bind to its receptors with high or low affinity (Trk A or p75NTR), and regulate series of vital processes in nerve cells such as survival, multiplication, differentiation, synaptic growth and apoptosis, exhibiting a promising clinical application perspective. In the previous study, we had screened out two peptides (NGF9/NGF12) that could bind to p75NTR and mimic anti-apoptosis function of NGF on R2L1 cultured with depriving serum medium by utilizing established R2L1 cell model which expressed p75NTR and phage display peptide library.Based on the previous studies, our research focuses on obtaining the fusion protein of EpoAB-NGF9/12 and testing its biological activity in vitro and in vivo. The following major work has been done:1. Constructed prokaryotic plasmids which can express GST-EpoAB-NGF9/12 in E.coli.;2. Optimized conditions of protein expression and purification of GST-EpoAB-NGF9/12 with high yield (about 40 mg/L) and high purity (about 90%);3. Verified NGF-like biological activity of GST-EpoAB-NGF9/12 in vitro on two kinds of cell line, tested neurite outgrowth induction activity of on PC 12 cell line and anti-apoptosis fuction on R2L1 cell model cultured with depriving serum medium;4. Attempted to establish EAE animal model on C57BL/6 mice and treat them with Epo and EpoAB, and obtained much experience for the further study on biological activity of EpoAB-NGF9/12 in vivo. Our study above has layed a foundation for the further study on blood-brain barrier permeability, nerval protective/nutritional pluripotency and mechanism of EpoAB-NGF9/12, and will benefit the clinical therapy of nerve injury, neurodegenerative disease in animal model or treatment for relevant human diseases.