The Reorganization Epoab-of Ngf Mimetic Peptide Expression And Purification Of The Preliminary Studies

Erythropoietin and recombinant erythropoietin has been applied in curing different kinds of anaemia clinically for a long time.It was newly found EPO having cytoprotective and nutritional effects in neural system and blood vascular system which does not depend on erythropoiesis.Moreover,its nerval protective and nutritional action has been positioned in a polypeptide sequence composed by seventeen amino acids which are AEHCSLNENITVPDTKV and called Epo-AB peptide.Some research has make it clear that when Epo-AB peptide performs the nerval protective and nutritional action,it has no erythropoiesis effect.In addition,Epo can permeate the brain blood barrier.So,Epo-AB has a very nice applicaton perspective.The nerve growth factor family includes NGF,BDNK NT-3,NT-4/5,and so on.They could regulate series of vital process such as survival,multiplication,differentiation, synapse growth and apoptosis of neurons by their binding to Trk or p75NTR with high or low affinity.Though NGF may has a good application perspective,it is not able to pass the blood brain barrier for its large molecular weight.While my laboratory has constructed an apoptosis cell model which can express p75NTR.Then got mimic peptides of NGF via the cell model and phage display technology.Those mimic peptide have a small molecular weight and may pass the blood brain barrier to play function after fused with other related proteins.The thesis topic is to express single EpoAB peptide and a fusion protein of EpoAB-NGF mimic peptide by constructing fusion gene and Pichia pastoris expression system.Then to explore the suitable expression and purification condition of the recombinant proteins.Above mentioned work will be the basis of further research and clinical application of the products.Firstly,related gene fragments were chemically synthesized,and overlapping PCR was performed to get the fusion gene sequence "EcoRI-α-factor-EGF-thrombin site-Epo AB-ligation peptide-EcoRI" and "EcoRI-α-factor-Epo AB-EcoRI".Then they were cloned into pUC18.Secondly,using the recombinant pUC18 as a template,the coding sequences for NGF 9 or 12 amino acids mimic peptide were inserted into the appropriate site by overlapping PCR and the DNA fragment "EcoRI-α-factor-EGF-thrombin site-Epo AB-ligation peptide-NGF 9 or 12 amino acids mimic peptide -EcoRI" was cloned into pUC 18 vector again.EcoRI endonuclease was used to get the fusion gene from recombinant vector and to cut the pAO815 plasmid.The fusion gene was ligated into the digested vector pAO815 to obtain an expression vector.The multicopy plasmids were contructed by using BamHI and BglII enzyme cutting recombinant pAO815 to get single expression cassettes consisted of 5'AOX promoter- fusion gene-3'AOX terminal sequence,and ligating the cassettes itself to get multi-copy expression cassette and inserting the multy cassettes into the BamHI site of the recombinant pAO815.The hexa-copy expression plasmid was constructed and transformed into Pichia pastoria by electrotransformation.The recombinant protein was induced and purified to a high level by using hydrophobic interaction chromatography,ion exchange chromatography and gel filtration chromatography.Two kinds of positive proteins were analyzed by western blotting with EGF antibody, spectrogram,peptide map and bioinformatics tools.