【作者】 史慧玲；

【导师】 王天厚；

【作者基本信息】 华东师范大学 ， 生态学， 2012， 博士

【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。更多还原

【Abstract】 Virus is a kind of tiny microorganism with simple no-cell structure and nucleotides (DNA or RNA). It has to parasitize in living cells and reproduces in the way of replication. Virus can be classified by shape and form as, spherical virus, baculovirus, Brick shaped virus, coronavirus, filamentous virus, spherical virus with envelop and virus with globular domain.Coronavirus and Influenza virus A have pathogenicity to human and animals. Most of the recent research about virus was focused on intracellular gene therapy, especially on Congenital host innate immunity However the pathogenicity mechanisms of many viruses are currently unknown. So the development of vaccines and gene therapy faced many difficulties. In this thesis, to clarify the feasibility of new development, we processed some research on pathogenicity of coronavirus Nspl protein, polyclonal antibody of HR2domain and monoclonal antibodies of influenza A virus M2e protein.Coronaviruses are divided into three groups based on serological criteria. Some members of the group II coronaviruses encode a number of proteins that antagonize host innate immunity. Nspl proteins are a major virulence and pathogenicity factor. The Nspl proteins of SARS-CoV promote host mRNA degradation and suppress host gene expressionTo under the pathogenicity of coronavirus Nspl protein, we compared the model of Nspl protein from group I coronavirus HCoV-229E, HCoV-NL63and group Ⅱ coronavirus SARS-CoV. We found they are common in folding ways and have similar structure and function. We made analysis the interaction between Nspl protein and cellar protein by immune-blotting and Co-IP. The result indicated Nspl protein from group I coronavirus HCoV-229E and HCoV-NL63could bind with ribosomal40S subunit to suppress host cell protein synthesis. Then we found IRF-3phosphorylation was not inhibited by Nsp1proteins after the analysis about the innate immune signal transduction which was stimulated by NDV infection in cells transfected with plasmids-expressing Nspl from HCoV-229E, HCoV-NL63, SARS-CoV control plasmid. The real time PCR results indicated that Nspl proteins could inhibit IFN-β and Luciferase mRNA transcription. And we found there was greater suppression of IFN-β and luciferase proteins synthesis by Nspl proteins in host cells infected by low pathogenic coronavirus than by SARS-CoV. All these results indicate that different coronaviruses might employ the same Nspl mechanism to antagonize host innate immunity and cell proliferation. However, Nspl may not be the key determinant of viral pathogenicity, or the factor used by the SARS coronavirus to evade host innate immunity.To develop new coronavirus vaccines, we analyzed the immunogenicity of HCoV-229E and HCoV-NL63using real-time PCR and western blotting. We found th at HCoV-229E has high immunogenicity and it could be made to useful coronavirus v accines.Antiviral strategies against HCoVs have been remarkably updated in recent years, especially with the emergence of the SARS-CoV. The HR region in coronavirus Spike glycoprotein is a promising therapeutic target.To investigate the Immunogenicity and the Neutralizing activity of HR region, we find out the amino acid sequences of five known coronavirus spike glycoprotein. Multiple alignments of the five HCoV HR2regions from different serogroups indicate that this region is relatively conserved. We purified the Spike fragments covering the entire HR2domain and the immediate upstream region for the five known HCoVs. After six times injection in mice, we get the five polyclonal antibodies. By the cross neutralization profile of the five anti-HR2polyclonal antibodies, we found the antibodies neutralize their antigenic virus or pseudo particles except the HCoV-HKU antibodies. To evaluate the specificity of the five HCoVs anti-HR2polyclonal antibodies, we performed Western blotting and found four of them were cross-reactive with other S proteins of the same or different serogroups. Moreover, we compared the amino acid sequence alignment in the HR2and upstream region from TGEV HR2and found it highly conserved. The neutralization assay showed the anti-TGEV-HR2antibodies have neutralization abilities with HCoV-NL63and HCoV-229E. Our data indicated the HR2and upstream regions in coronavirus spike (S) glycoprotein is a promising therapeutic target.Our results make deeper understanding on mechanisms of virus pathogenicity and bring new insight in developing vaccine and therapeutic agents against virus infections.更多还原