重组EpoAB-NGF模拟肽在大肠杆菌中的表达、纯化及生物活性初步研究

【作者】 潘勇昭；

【导师】 陈虹；

【作者基本信息】 北京协和医学院 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 红细胞生成素(Epo)最先作为一个促进红细胞生成的生长因子被确认,后来发现其在神经、血管等系统中具有不依赖于红细胞生成作用的细胞营养保护功能,并且这种营养保护作用已经被定位到了其序列中一个由17个氨基酸组成的多肽顺序上,称作EpoAB肽(AEHCSLNENITVPDTKV)。神经生长因子(NGF)可通过结合靶细胞膜上的高/低亲和力受体(Trk/p75NTR)调控神经元的存活、增殖、分化、突触生长和凋亡等生命过程,有较好的临床应用前景。本室利用构建的表达p75NTR的R2L1凋亡细胞模型和噬菌体展示文库筛选出2个有抗凋亡作用的p75NTR的模拟配基,即NGF的模拟肽(NGF9和NGF12)。本研究在前期工作的基础上,构建了原核表达GST-EpoAB-NGF9/12融合蛋白的重组质粒,筛选相应的表达菌株,优化了表达和纯化条件,获得融合蛋白,并通过刺激PC12细胞轴突生长和抑制R2L1细胞去血清凋亡的实验,初步证实了融合蛋白对神经细胞的营养保护作用。另外,尝试建立了EAE动物模型并用Epo和EpoAB进行了初步治疗,为进一步验证EpoAB-NGF9/12在动物体内的活性积累了经验。本研究工作的开展,可为日后研究该重组蛋白的血脑屏障通透性,神经营养保护作用的多能性和可能的作用机制等打下基础,进而探索其在神经损伤、神经退行性病变动物模型或人类相应疾病的临床治疗上的应用前景。更多还原

【Abstract】 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.更多还原