六种抗大鼠Nogo-A分子单克隆抗体的免疫组织化学染色特性及Nogo-A分子截短体的构建

发布时间：2018-02-25 01:20

本文关键词： Nogo-A 单克隆抗体免疫荧光组织化学截短体融合蛋白抗原表位　出处：《第四军医大学》2011年硕士论文　论文类型：学位论文

【摘要】：成年哺乳动物中枢神经系统(CNS)损伤后,由于神经再生的困难致使其结构和功能的恢复非常有限。神经元自身再生能力的低下以及损伤后不利于再生的内环境是造成中枢神经系统损伤愈后不良的主要原因。最近20年,人们先后发现了多种髓鞘源性的抑制蛋白,包括Nogo-A、髓鞘碱性蛋白MAG、少突胶质细胞髓磷脂糖蛋白OMgp、轴突导向因子netrins等。特别是对Nogo-A、Nogo-A受体NgR及Nogo-A受体复合物(p75~(NTR)/NgR1/LINGO-1及TROY/NgR1/LINGO-1)的结构、定位和功能进行了细致而深入的探讨,分析了它们在CNS损伤修复过程中扮演的角色,为解决CNS损伤修复提供了新的治疗靶点。新近研究发现,Nogo-A及其受体复合物在CNS发育的不同阶段对神经前体细胞迁移以及神经突触生长和可塑性等具有调控作用;对少突胶质细胞分化和髓鞘化具有促进作用;在CNS损伤后炎性细胞的迁移运动以及多种神经免疫性疾病中发挥一定作用。-4-这些预示着Nogo-A及其受体复合物功能的多样性,还有更广阔的研究空间和重要的研究价值。以往研究表明,轴突生长抑制因子Nogo-A具有抑制CNS损伤后轴突的生长和分枝形成的作用。目前已经明确Nogo-A的三个抑制性区域:一个是位于C端的Nogo-66(aa 1026-1091)区域,另外两个是分别位于N端的aa 1-172和aa 174-979区域。通过给予针对Nogo-A分子的功能阻断性抗体、NgR拮抗剂、可溶性NgR片段等方法,阻断Nogo-A与其受体结合,抑制下游信号途径,从而可以显著促进CNS损伤后轴突的再生和运动功能的恢复。然而,由于目前商品化的抗Nogo-A抗体种类比较少,功能比较单一,极大的影响了对Nogo-A分子功能的深入研究,特别是其在CNS损伤修复的相关研究中显得尤为突出。虽然部分实验证明通过功能封闭性抗体抑制Nogo-A的功能可以显著促进轴突再生,但其具体的作用靶点和短肽药物还没有明确和研制生产,从基础研究到临床应用的转化还有很多未知需要不断深入的探索下去。我们前期通过原核表达大鼠Nogo-A分子氨基端(Amino-Nogo-A)的aa 570-691片段和Nogo-A分子羧基端(Nogo-66)aa 1026-1091片段,通过免疫BALB/c-nude~(-/-)小鼠,获得6株针对Nogo-A分子的单克隆抗体,分别命名为Nogo66-1 mAb,Nogo66-2 mAb,Nogo66-3 mAb,Nogo66-4 mAb,NogoN-1 mAb,NogoN-2 mAb。Western blot检测可以识别健康大鼠Nogo-A蛋白(200 KD)。但这些抗体是否可以识别中枢神经系统中的Nogo-A分子还不得而知,这极大的影响了上述6种抗体的应用。我们想通过鉴定6种Nogo-A单克隆抗体的免疫组织化学特性及构建针对Nogo-A分子抑制性区域的不同截短体,为下一步研究抗体的功能特点、作用表位、相关信号途径及具体应用奠定基础。实验第一部分,我们制备了6种抗Nogo-A单克隆抗体的腹水,应用免疫荧光组织化学方法,比较这6种Nogo-A单抗在大鼠脊髓组织中免疫荧光组织化学染色情况,检测其对大鼠神经组织的反应性与特异性,以便了解各个抗体与组织中Nogo-A分子的结合能力以及相关抗原在神经细胞的分布情况,为有效应用上述抗体打下基础。实验第二部分,我们针对Nogo-A分子氨基端(Amino-Nogo-A)的aa 570-691区域和Nogo-A分子羧基端(Nogo-66)aa 1026-1091区域,通过RT-PCR技术获得不同长度的截短体片段,通过质粒重组方法,构建带有GST标签的不同截短体的重组质粒,通过融合蛋白的原核表达和Western blot技术,进一步鉴定6种Nogo-A分子单克隆抗体作用的片段,为下一步研究该抗体的功能活性及在中枢神经系统损伤修复、炎性调控等作用提供实验资源。通过以上两部分实验,我们得到如下结果: 1.正常SD大鼠脊髓组织冠状切面的免疫荧光组织化学双标结果显示,制备的6种抗大鼠Nogo-A分子的单克隆抗体(mAb)均可以与商品化的兔抗大鼠Nogo-A多克隆抗体(pAb)双标记; 2. Nogo66-1、Nogo66-2、Nogo66-4和NogoN-2 mAb可与MBP阳性细胞双标记,与GFAP阳性细胞不共存;而Nogo66-3和NogoN-1 mAb不仅可以与MBP阳性细胞双标,同时也可以和GFAP阳性细胞共存。 3.我们成功的将含有Nogo-66(aa 1026-1091)和Amino-Nogo-A(aa 570-691)的不同区域的片段亚克隆至pGEX-4T1的载体中,构建了多个重组质粒;通过融合蛋白原核表达方法,使带GST标签的各个截短体在原核表达系统中成功表达,得到GST-△Nogo66_a(aa1026-1091)、GST-△Nogo66_b(aa1056-1091)、GST-△NogoA-N_a(aa 570-691)、GST-△NogoA-N_b(aa 601-691)、GST-△NogoA-N_c(aa 634-691)、GST-△NogoA-N_d(aa 669-691)的融合蛋白。 4.通过Western blot技术,鉴定了6种Nogo-A分子单克隆抗体结合的肽段位置,即Nogo66-1 mAb、Nogo66-2 mAb、Nogo66-3 mAb、Nogo66-4 mAb所识别的片段位于aa 1026-1055中;NogoA-N1 mAb、NogoA-N2 mAb所识别的表位在aa 634-668的位置。由以上实验,我们认为,Nogo66-1、Nogo66-2、Nogo66-4和NogoN-2 mAbs可很好识别脊髓组织中的Nogo-A分子,适用于免疫组化研究;Nogo66-1、Nogo66-2、Nogo66-3、Nogo66-4 mAbs针对Nogo-A的aa 1026-1055片段,NogoA-N-1、NogoA-N-2 mAbs针对Nogo-A的aa 634-668片段。通过以上研究,明确了6种抗Nogo-A分子单克隆抗体的免疫荧光组织化学的特性及成功的构建了针对Nogo-A分子抑制性区域的不同截短体,初步鉴定了6种NogoA单克隆抗体识别的具体肽段,为有效应用上述抗体打下了坚实基础。
[Abstract]:The adult mammalian central nervous system (CNS) after injury due to nerve regeneration. So the structure and function of the recovery is very limited. The neurons in the regeneration after injury is low and not conducive to environmental regeneration is mainly caused by the injury of central nervous system after bad.
The last 20 years, people have found a variety of myelin derived proteins, including Nogo-A, myelin basic protein MAG, oligodendrocyte myelin glycoprotein OMgp, axon guidance factor netrins. Especially for Nogo-A, Nogo-A receptor NgR and Nogo-A receptor complex (p75~ (NTR) /NgR1/LINGO-1 and TROY/NgR1/LINGO-1) of the structure, positioning and the function of detailed and in-depth analysis of the process, they play a role in CNS damage repair, provides a new therapeutic target for CNS damage repair. Recent studies showed that Nogo-A and its receptor complexes at different stages of CNS development of neural precursor cell migration and synaptic growth and plasticity has a regulatory role; plays a promoting role in oligodendrocyte differentiation and myelination in glial cells; CNS damage after the migration of inflammatory cells and a variety of neurological autoimmune diseases play a The action of.-4- indicates the diversity of the function of Nogo-A and its receptor complex, and there is a broader research space and important research value.
Previous studies showed that axon growth inhibitory factor Nogo-A can inhibit axonal growth and branch formation damage after CNS. It is now clear that Nogo-A three inhibitory regions: one is located in the C terminal Nogo-66 (AA 1026-1091), the other two are located in the N end of AA 1-172 and AA 174-979 regions. By giving to Nogo-A molecular function blocking antibody, NgR antagonists, soluble NgR fragments and other methods, blocking the binding of Nogo-A to its receptors, inhibit downstream signaling pathways, which can significantly promote axonal regeneration and motor function after CNS injury recovery.
However, due to the current anti Nogo-A antibody species commercialization is relatively small, single function, greatly affected the further research on Nogo-A molecular function, especially the damage repair related research is particularly prominent in CNS. Although some experiments show that through the function blocking antibody can inhibit the function of Nogo-A can significantly promote axon regeneration. The target and peptide drugs have not been identified and developed, translation from basic research to clinical application there are many questions to explore.
We first determined by prokaryotic expression of rat Nogo-A molecule amino terminal (Amino-Nogo-A) of the 570-691 AA fragment and Nogo-A molecular C-terminal (Nogo-66) AA 1026-1091 fragment by immune BALB/c-nude~ (- / -) mice, 6 strains of monoclonal antibodies against Nogo-A molecules, which were named as Nogo66-1 mAb, Nogo66-2 mAb, Nogo66-3 mAb, Nogo66-4 mAb. NogoN-1 mAb, NogoN-2 mAb.Western blot can detect Nogo-A protein identification in healthy rats (200 KD). But whether these antibodies can identify Nogo-A molecule in the central nervous system also can make nothing of it, which has a great influence on the application of the above 6 kind of antibody. And we want to construct different truncated inhibitory area for Nogo-A molecules in immune tissues chemical characterization of 6 kinds of monoclonal antibodies against Nogo-A, as a function of the antibody in the next step, the role of epitope, lay the foundation of related signal pathway and the specific application.
The first part of the experiment, we prepared 6 kinds of monoclonal antibodies against Nogo-A ascites by immunofluorescence method, comparing the 6 Nogo-A monoclonal antibody in rat spinal cord tissue immunofluorescence staining, to detect the rat nerve tissue reactivity and specificity, in order to understand the molecular binding ability of each Nogo-A antibody and related antigens in tissues and the distribution of nerve cells, and lay the foundation for the effective application of the antibodies.
In the second part, we aimed at the N-terminal Nogo-A molecule (Amino-Nogo-A) AA 570-691 Nogo-A molecules and carboxyl terminal region (Nogo-66) AA 1026-1091, obtained truncated fragments of different lengths by RT-PCR technology, through the method of recombinant plasmid, recombinant plasmid of different truncated GST tagged fusion protein, the prokaryotic expression Western and blot technology, further identification of 6 Nogo-A molecular effect of monoclonal antibody fragments for further study of the antibody activity and function in the central nervous system injury, inflammatory regulation effects and provide experimental resources.
Through the two parts of the experiment, we get the following results:
1. the immunofluorescence histochemistry double labeled results of normal SD rat spinal coronal section showed that the 6 monoclonal antibodies (mAb) against rat Nogo-A molecule could be labeled with commercial Rabbit anti Nogo-A polyclonal antibody (pAb).
2. Nogo66-1, Nogo66-2, Nogo66-4 and NogoN-2 mAb can be double labeled with MBP positive cells, but do not coexist with GFAP positive cells. Nogo66-3 and NogoN-1 mAb can not only be double labeled with the positive cells, but also coexist with the positive cells.
3. of our success with Nogo-66 (AA 1026-1091) and Amino-Nogo-A (AA 570-691) in different regions of the vector fragment was subcloned to pGEX-4T1, constructed several recombinant plasmid; prokaryotic expression by the method, so that each truncated GST tag was successfully expressed in prokaryotic expression system, get GST- Nogo66_a (aa1026-1091), GST- Nogo66_b (aa1056-1091), GST- NogoA-N_a (AA 570-691), GST- NogoA-N_b (AA 601-691), GST- NogoA-N_c (AA 634-691), GST- NogoA-N_d (AA 669-691) of the fusion protein.
4., through Western blot technology, we identified 6 peptide binding sites of Nogo-A molecule monoclonal antibody, that is, Nogo66-1 mAb, Nogo66-2 mAb, Nogo66-3 mAb, Nogo66-4 mAb, and the identified fragments were located in the Nogo66-4 1026-1055. The location of the identified epitopes was 634-668.
From the above experiments, we believe that Nogo66-1, Nogo66-2, Nogo66-4 and NogoN-2 mAbs Nogo-A can be good molecular recognition in spinal cord tissue, suitable for immunohistochemical study; Nogo66-1, Nogo66-2, Nogo66-3, Nogo66-4 mAbs for Nogo-A AA 1026-1055 NogoA-N-1 NogoA-N-2 mAbs fragment for Nogo-A 634-668 AA fragments. Through the above research, clear 6 kinds of monoclonal anti Nogo-A antibody molecules immunofluorescence histochemical characteristics and construct different truncated inhibitory region for Nogo-A molecules, identification of specific peptide 6 NogoA monoclonal antibodies, laid a solid foundation for the effective application of the antibody.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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