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| Preliminary Study on Neurotrophic Activity of Recombinant Peptide Mimics EpoAB-NGF |
| PAN Yong-zhao, CHEN Hong, WANG Xin, MA Xiao-li, HUANG Bing-ren |
| National Laboratory of Medical Molecular Biology, Institute of Basic Medical Science, CAMS and PUMC, Beijing 100005, China |
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Abstract 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.
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Received: 06 September 2010
Published: 25 January 2011
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