發(fā)布時間：2018-02-25 01:20

  本文關鍵詞： Nogo-A 單克隆抗體 免疫熒光組織化學 截短體 融合蛋白 抗原表位　出處：《第四軍醫(yī)大學》2011年碩士論文　論文類型：學位論文

【摘要】：成年哺乳動物中樞神經系統(tǒng)(CNS)損傷后,由于神經再生的困難致使其結構和功能的恢復非常有限。神經元自身再生能力的低下以及損傷后不利于再生的內環(huán)境是造成中樞神經系統(tǒng)損傷愈后不良的主要原因。 最近20年,人們先后發(fā)現了多種髓鞘源性的抑制蛋白,包括Nogo-A、髓鞘堿性蛋白MAG、少突膠質細胞髓磷脂糖蛋白OMgp、軸突導向因子netrins等。特別是對Nogo-A、Nogo-A受體NgR及Nogo-A受體復合物(p75~(NTR)/NgR1/LINGO-1及TROY/NgR1/LINGO-1)的結構、定位和功能進行了細致而深入的探討,分析了它們在CNS損傷修復過程中扮演的角色,為解決CNS損傷修復提供了新的治療靶點。新近研究發(fā)現,Nogo-A及其受體復合物在CNS發(fā)育的不同階段對神經前體細胞遷移以及神經突觸生長和可塑性等具有調控作用;對少突膠質細胞分化和髓鞘化具有促進作用;在CNS損傷后炎性細胞的遷移運動以及多種神經免疫性疾病中發(fā)揮一定作用。-4-這些預示著Nogo-A及其受體復合物功能的多樣性,還有更廣闊的研究空間和重要的研究價值。 以往研究表明,軸突生長抑制因子Nogo-A具有抑制CNS損傷后軸突的生長和分枝形成的作用。目前已經明確Nogo-A的三個抑制性區(qū)域:一個是位于C端的Nogo-66(aa 1026-1091)區(qū)域,另外兩個是分別位于N端的aa 1-172和aa 174-979區(qū)域。通過給予針對Nogo-A分子的功能阻斷性抗體、NgR拮抗劑、可溶性NgR片段等方法,阻斷Nogo-A與其受體結合,抑制下游信號途徑,從而可以顯著促進CNS損傷后軸突的再生和運動功能的恢復。 然而,由于目前商品化的抗Nogo-A抗體種類比較少,功能比較單一,極大的影響了對Nogo-A分子功能的深入研究,特別是其在CNS損傷修復的相關研究中顯得尤為突出。雖然部分實驗證明通過功能封閉性抗體抑制Nogo-A的功能可以顯著促進軸突再生,但其具體的作用靶點和短肽藥物還沒有明確和研制生產,從基礎研究到臨床應用的轉化還有很多未知需要不斷深入的探索下去。 我們前期通過原核表達大鼠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,通過免疫BALB/c-nude~(-/-)小鼠,獲得6株針對Nogo-A分子的單克隆抗體,分別命名為Nogo66-1 mAb,Nogo66-2 mAb,Nogo66-3 mAb,Nogo66-4 mAb,NogoN-1 mAb,NogoN-2 mAb。Western blot檢測可以識別健康大鼠Nogo-A蛋白(200 KD)。但這些抗體是否可以識別中樞神經系統(tǒng)中的Nogo-A分子還不得而知,這極大的影響了上述6種抗體的應用。我們想通過鑒定6種Nogo-A單克隆抗體的免疫組織化學特性及構建針對Nogo-A分子抑制性區(qū)域的不同截短體,為下一步研究抗體的功能特點、作用表位、相關信號途徑及具體應用奠定基礎。 實驗第一部分,我們制備了6種抗Nogo-A單克隆抗體的腹水,應用免疫熒光組織化學方法,比較這6種Nogo-A單抗在大鼠脊髓組織中免疫熒光組織化學染色情況,檢測其對大鼠神經組織的反應性與特異性,以便了解各個抗體與組織中Nogo-A分子的結合能力以及相關抗原在神經細胞的分布情況,為有效應用上述抗體打下基礎。 實驗第二部分,我們針對Nogo-A分子氨基端(Amino-Nogo-A)的aa 570-691區(qū)域和Nogo-A分子羧基端(Nogo-66)aa 1026-1091區(qū)域,通過RT-PCR技術獲得不同長度的截短體片段,通過質粒重組方法,構建帶有GST標簽的不同截短體的重組質粒,通過融合蛋白的原核表達和Western blot技術,進一步鑒定6種Nogo-A分子單克隆抗體作用的片段,為下一步研究該抗體的功能活性及在中樞神經系統(tǒng)損傷修復、炎性調控等作用提供實驗資源。 通過以上兩部分實驗,我們得到如下結果: 1.正常SD大鼠脊髓組織冠狀切面的免疫熒光組織化學雙標結果顯示,制備的6種抗大鼠Nogo-A分子的單克隆抗體(mAb)均可以與商品化的兔抗大鼠Nogo-A多克隆抗體(pAb)雙標記; 2. Nogo66-1、Nogo66-2、Nogo66-4和NogoN-2 mAb可與MBP陽性細胞雙標記,與GFAP陽性細胞不共存;而Nogo66-3和NogoN-1 mAb不僅可以與MBP陽性細胞雙標,同時也可以和GFAP陽性細胞共存。 3.我們成功的將含有Nogo-66(aa 1026-1091)和Amino-Nogo-A(aa 570-691)的不同區(qū)域的片段亞克隆至pGEX-4T1的載體中,構建了多個重組質粒;通過融合蛋白原核表達方法,使帶GST標簽的各個截短體在原核表達系統(tǒng)中成功表達,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.通過Western blot技術,鑒定了6種Nogo-A分子單克隆抗體結合的肽段位置,即Nogo66-1 mAb、Nogo66-2 mAb、Nogo66-3 mAb、Nogo66-4 mAb所識別的片段位于aa 1026-1055中;NogoA-N1 mAb、NogoA-N2 mAb所識別的表位在aa 634-668的位置。 由以上實驗,我們認為,Nogo66-1、Nogo66-2、Nogo66-4和NogoN-2 mAbs可很好識別脊髓組織中的Nogo-A分子,適用于免疫組化研究;Nogo66-1、Nogo66-2、Nogo66-3、Nogo66-4 mAbs針對Nogo-A的aa 1026-1055片段,NogoA-N-1、NogoA-N-2 mAbs針對Nogo-A的aa 634-668片段。通過以上研究,明確了6種抗Nogo-A分子單克隆抗體的免疫熒光組織化學的特性及成功的構建了針對Nogo-A分子抑制性區(qū)域的不同截短體,初步鑒定了6種NogoA單克隆抗體識別的具體肽段,為有效應用上述抗體打下了堅實基礎。
[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.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392.1

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相關期刊論文 前1條

1 程希平;劉惠玲;宋朝君;金伯泉;焦西英;游思維;鞠躬;;Nogo-A在成年大鼠脊髓和背根節(jié)的分布[J];Neuroscience Bulletin;2005年01期

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本文編號：1532469