冠状病毒Nsp1蛋白生物学机理研究及HR2片段的抗体研发

发布时间：2018-01-14 13:20

本文关键词：冠状病毒Nsp1蛋白生物学机理研究及HR2片段的抗体研发　出处：《华东师范大学》2012年博士论文　论文类型：学位论文

【摘要】：病毒是一类个体微小,结构简单,只含单一核酸(DNA或RNA),必须在活细胞内寄生并以复制方式增殖的非细胞型微生物。根据病毒的形态分类可分为球状病毒,杆状病毒,砖形病毒,冠状病毒,丝状病毒,有包膜的球状病毒和具有球状头部的病毒等。在众多动物病毒中,冠状病毒和甲型流感病毒都是一类对人类和动物一定致病性的病原体。目前对于病毒的主要研究热点集中于病毒细胞内基因治疗,特别是针对宿主细胞先天性天然免疫方面[62],然而由于对于许多病毒的致病机制研究尚不完全清楚,针对性的疫苗研发和细胞内基因治疗也一直而临许多困难。本文围绕冠状病毒Nsp1蛋白的致病性,HR2区域多克隆抗体研发及甲型流感病毒M2e蛋白单克隆抗体研发展开研究,阐述了它们应用于新型的细胞内基因治疗及疫苗研发的可行性。冠状病毒依照不同血清群体而分为三大类,第二类冠状病毒中某些物种能够编码一些蛋白抵御宿主细胞先天性天然免疫反应。其中非结构蛋白Nsp1是冠状病毒的主要致病因子之一。有研究表明SARS-CoV的非结构蛋白Nsp1可以促进宿主细胞mRNA的降解和宿主细胞基因的表达[46,61,83]。为了研究冠状病毒Nsp1蛋白的致病性,我们将第一类冠状病毒HCoV-229E及HCoV-NL63与第二类冠状病毒SARS-CoV的非结构蛋白Nsp1的模型进行对比试验,发现HCoV-229E和HCoV-NL63的非结构蛋白Nsp1的折叠方式和SARS-CoV的非结构蛋白Nsp1的折叠方式极为相似,并且这些蛋白有着相似的结构和功能。我们利用免疫印迹反应和免疫共沉淀(Co-IP)的方法分析非结构蛋白Nspl与细胞蛋白之间的相互作用关系,结果显示第一类冠状病毒HCoV-229E和HCoV-NL63的非结构蛋白Nsp1可以结合核糖体亚基40S,抑制宿主免疫与非免疫蛋白的合成。接着我们利用细胞转染和病毒激活技术,发现非结构蛋白Nsp1不阻止IRF3的磷酸化。我们利用实时定量PCR的方法,研究表明非结构蛋白Nsp1可以抑制干扰素-p(IFN-p)及荧光素酶的mRNA的转录。然后根据荧光素酶报告分析表明非结构蛋白NSP1对低致病性冠状病毒的宿主细胞蛋白的合成的影响要强于对SARS-CoV病毒的影响。这些结果表明不同的冠状病毒可能都可以利用非结构蛋白Nsp1来抵御宿主先天性天然免疫反应和宿主细胞的增殖,然而非结构蛋白Nsp1可能并不是病毒致病性因子中的决定性因子。为了开发研究针对冠状病毒的疫苗,我们选择.了HCoV-229E和HCoV-NL63这两种人类常见病毒,并利用免疫印迹反应,实时定量PCR和ELISA实验对它们进行了免疫原性的分析,结果表明HCoV-229E病毒具有极高的免疫原性,并满足研发成为病毒疫苗的基本条件。 SARS-CoV出现以后,关于冠状病毒的抗病毒治疗方而的研究越来越多。冠状病毒Spike刺突糖蛋白的HR2(Heptad Repeat, HR)片段已被证明是有希望用来进行细胞内基因治疗的重要靶位点[116,118]。为了研究HR区域的免疫原性和中和活性,我们首先对五种已知的冠状病毒Spike刺突糖蛋白的氨基酸片段进行同源性分析,找出HR2区域及相邻上游区域片段的同源片段序列。接着我们纯化出HR2区域及相邻上游区域片段的蛋白,对老鼠进行免疫注射并得到多克隆抗体。通过利用多克隆抗体进行抗体交叉中和活性检测,我们发现除了HCoV-HKU1病毒以外,其他四种针对HR2区域及相邻上游区域的抗体均表现出针对同类型病毒或者假病毒的抗体交叉中和活性。接下来我们又进行了针对五种冠状病毒的Spike刺突蛋白的免疫印迹反应实验,结果显示五种人类冠状病毒中的四种抗HR2的抗体能够与来自相同或者不同血清群的冠状病毒的Spike蛋白发生交叉反应。此外,我们又对猪肠道病毒(TGEV)的HR2区域及相邻上游区域进行了分析,发现该区域有着极高的保守性,并对该区域进行免疫印迹反应实验和抗体交叉中和活性检测,结果证明针对TGEV病毒的HR2区域及相邻上游区域的抗体检测到与HCoV-NL63和HCoV-229E病毒的交叉反应和抗体中和活性。我们的研究结果表明冠状病毒的HR2区域及相邻上游区域片段是在未来针对已知冠状病毒或者可能出现新型冠状病毒在疫苗研发或者细胞内基因治疗方面的重要研究靶位点。综上所述,本文中的结果填补了冠状病毒致病机理研究上的一些空白,促进了关于冠状病毒疫苗研发及抗病毒治疗方法的研究。
[Abstract]:Virus is a kind of individual small, simple structure, containing only a single nucleic acid (DNA or RNA), must be parasitic in living cells and to copy the way the proliferation of non cellular microorganisms. According to the morphological classification of the virus can be divided into spherical virus, baculovirus, brick shaped virus, coronavirus, filamentous virus, envelope globular the head of the virus and has a spherical virus.
In a number of animal virus in the coronavirus and influenza A virus is a kind of human and animal pathogenic pathogens. Some current research focus for the virus in virus cell in gene therapy, especially for the host cell natural immune [62], however the study of pathogenic mechanism for many viruses is not completely clearly, targeted vaccine development and gene therapy has also been faced with many difficulties. This paper focuses on the pathogenicity of coronavirus Nsp1 protein, HR2 polyclonal antibody and influenza a M2e influenza virus research monoclonal antibody research and development research, expounds their application to new cells for gene therapy and vaccine development feasibility.
According to the different groups of serum coronavirus is divided into three categories, second kinds of coronavirus in some species can resist host cell protein encoding some congenital innate immune reaction. The non structural protein Nsp1 is one of the main pathogenic factor of coronavirus. Studies have shown that SARS-CoV can promote the expression of non structural protein Nsp1 of [46,61,83]. degradation and host host cell mRNA cell gene
In order to study the pathogenicity of coronavirus Nsp1 protein, we will be the first class of HCoV-229E and HCoV-NL63 and the second type of coronavirus SARS-CoV coronavirus nonstructural protein Nsp1 model were compared, found that HCoV-229E and HCoV-NL63 fold non structural protein Nsp1 fold and SARS-CoV non structural protein Nsp1 is very similar, and these proteins are similar in structure and function. We used immunoblotting and immunoprecipitation (Co-IP) method to analyze the interaction between nonstructural protein Nspl and cell protein, showed the first class of coronavirus HCoV-229E and HCoV-NL63 non structural protein Nsp1 can bind to ribosomal subunit 40S, inhibit the synthesis of host immune and non immune proteins. Then we use the cell transfection and virus activation technology, found that non structural protein Nsp1 does not prevent the phosphorylation of IRF3. By using the method of real-time quantitative PCR, and studies show that non structural protein Nsp1 can inhibit interferon -p (IFN-p) transcription and luciferase mRNA. Then according to the luciferase reporter analysis showed that non structural protein NSP1 of low pathogenic coronavirus host cell protein synthesis effect is stronger than the effect on SARS-CoV virus. These results show that different may use coronavirus nonstructural protein Nsp1 to resist natural host innate immune responses and host cell proliferation, however, non structural protein Nsp1 is probably not the decisive factor in viral pathogenicity factor. In order to research and development of vaccines, for coronavirus we choose. HCoV-229E and HCoV-NL63 these two kinds of common human virus, and the use of the blot, real-time quantitative PCR and ELISA experiments were conducted to analyze the immunogenicity of them, the results showed that HCoV-229E disease Poison has a very high immunogenicity, and it meets the basic conditions of R & D to become a virus vaccine.
Since the advent of SARS-CoV, more and more researches have been done on the antiviral treatment of coronavirus. The HR2 (Heptad Repeat, HR) fragment of coronavirus Spike spike glycoprotein has been proved to be an important target site for gene therapy in cells.
To study the immunogenicity of HR region and neutralizing activity, we first amino acid fragment of the five known coronavirus spike glycoprotein Spike homology analysis of homologous fragment sequence, find the HR2 region and the adjacent region of upstream fragment. Then we purified HR2 region and the adjacent region upstream fragment of protein for immunization of mice and the polyclonal antibody. We conducted neutralization test cross antibody by using polyclonal antibody, we found that in addition to the HCoV-HKU1 virus, the other four for the HR2 area and the adjacent upstream region of the antibody showed antibodies to the same type of virus or false virus cross neutralization activity. We also conducted immunoblotting experiments for five Spike coronavirus spike protein, the results showed that five kinds of human coronavirus in four kinds of anti HR2 antibody and to From the same or different groups of serum coronavirus Spike protein cross react. In addition, we treat swine enterovirus (TGEV) in the HR2 region and its adjacent upstream region were analyzed, and found that the region has a highly conserved, and the area of Western blotting reaction experiments and cross neutralization test results antibody. Detection of HR2 antibody and the adjacent area that the upstream region for the TGEV virus to cross reaction and antibody neutralizing activity with HCoV-NL63 and HCoV-229E virus. The results of our study show that the HR2 region and adjacent coronavirus upstream region fragment is in the future for known coronavirus or may have important research target in the new coronavirus vaccine or cells the gene therapy sites.
To sum up, the results in this paper fill gaps in the pathogenesis of coronavirus, and promote the research of coronavirus vaccine development and antiviral therapy.

【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R373

【共引文献】