Preliminary Study on Neurotrophic Activity of Recombinant Peptide Mimics EpoAB-NGF

Objective:To investigate the neurotrophic activity of recombinant fusion protein epopeptide AB-nerve growth factor analog peptide(EpoAB-NGF9/12).Methods:The DNA fragments encoding the sequences of EpoAB-NGF9 and EpoAB-NGF12 polypeptide were amplified by PCR and cloned into the pET-42a prokaryotic expression vector.The recombinant plasmids of pET-42a-EpoAB-NGF9/12 were transformed into E.coli BL21(DE3) and exogenous protein expression was induced by IPTG.Fusion proteins GST-EpoAB-NGF9/12 were purified by affinity chromatography.The biological activities were determined using the experiment of inducing axon outgrowth of PC12 cells and flow cytometry analysis of apoptotic rate in R2L1 cells.Results:Apparent molecular weight of fusion proteins GST-EpoAB-NGF9/12 were approximate 30kDa.Immunoblot analysis showed that the fusion proteins were immunoreactive with anti-GST antibody.Fusion proteins stimulate the differentiation and promote the axon outgrowth in PC12 cells.Cell apoptosis was induced in(31.7±0.60)% of R2L1 control cells by serum free incubation,whereas cell apoptosis rate were in(25.2±3.52)% or(25.7±1.46)% by adding GST-EpoAB-NGF9 or GST-EpoAB-NGF12 into serum free condition respectively.The result indicates that fusion proteins were enabling to prevent cell death in R2L1 cells.Conclusion:These findings in cell biology region suggest that recombinant fusion protein containing Epo-NGF peptide mimics have the neurotrophic effects similar to that of NGF.