重组EpoAB-NGF模拟肽的神经营养功能

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

目的:研究重组融合蛋白红细胞生成素AB肽-神经生长因子模拟肽(EpoAB-NGF9和EpoAB-NGF/12)的神经营养作用。方法:构建pET-42a-EpoAB-NGF9/12原核表达质粒,转化大肠杆菌BL21(DE3),以IPTG诱导表达,亲和层析纯化重组蛋白,显微镜观察PC12细胞诱导分化,流式细胞仪检测凋亡细胞。结果:大肠杆菌表达的重组融合蛋白GST-EpoAB-NGF9/12分子量约为30kDa,抗GST抗体免疫印迹反应呈阳性。融合蛋白可以诱导PC12细胞分化,促进轴突生长。R2L1细胞去血清培养,凋亡细胞占(31.7±0.60)%,去血清后加入融合蛋白GST-EpoAB-NGF9/12,细胞凋亡率分别为(25.2±3.52)%、(25.7±1.46)%,呈现一定程度的抑制细胞凋亡的活性。结论:重组EpoAB-NGF模拟肽融合蛋白具有与NGF类似的神经营养作用。

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.