本文關鍵詞：冠狀病毒Nsp1蛋白生物學機理研究及HR2片段的抗體研發(fā)　出處：《華東師范大學》2012年博士論文　論文類型：學位論文

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【摘要】：病毒是一類個體微小,結構簡單,只含單一核酸(DNA或RNA),必須在活細胞內寄生并以復制方式增殖的非細胞型微生物。根據(jù)病毒的形態(tài)分類可分為球狀病毒,桿狀病毒,磚形病毒,冠狀病毒,絲狀病毒,有包膜的球狀病毒和具有球狀頭部的病毒等。 在眾多動物病毒中,冠狀病毒和甲型流感病毒都是一類對人類和動物一定致病性的病原體。目前對于病毒的主要研究熱點集中于病毒細胞內基因治療,特別是針對宿主細胞先天性天然免疫方面[62],然而由于對于許多病毒的致病機制研究尚不完全清楚,針對性的疫苗研發(fā)和細胞內基因治療也一直而臨許多困難。本文圍繞冠狀病毒Nsp1蛋白的致病性,HR2區(qū)域多克隆抗體研發(fā)及甲型流感病毒M2e蛋白單克隆抗體研發(fā)展開研究,闡述了它們應用于新型的細胞內基因治療及疫苗研發(fā)的可行性。 冠狀病毒依照不同血清群體而分為三大類,第二類冠狀病毒中某些物種能夠編碼一些蛋白抵御宿主細胞先天性天然免疫反應。其中非結構蛋白Nsp1是冠狀病毒的主要致病因子之一。有研究表明SARS-CoV的非結構蛋白Nsp1可以促進宿主細胞mRNA的降解和宿主細胞基因的表達[46,61,83]。 為了研究冠狀病毒Nsp1蛋白的致病性,我們將第一類冠狀病毒HCoV-229E及HCoV-NL63與第二類冠狀病毒SARS-CoV的非結構蛋白Nsp1的模型進行對比試驗,發(fā)現(xiàn)HCoV-229E和HCoV-NL63的非結構蛋白Nsp1的折疊方式和SARS-CoV的非結構蛋白Nsp1的折疊方式極為相似,并且這些蛋白有著相似的結構和功能。我們利用免疫印跡反應和免疫共沉淀(Co-IP)的方法分析非結構蛋白Nspl與細胞蛋白之間的相互作用關系,結果顯示第一類冠狀病毒HCoV-229E和HCoV-NL63的非結構蛋白Nsp1可以結合核糖體亞基40S,抑制宿主免疫與非免疫蛋白的合成。接著我們利用細胞轉染和病毒激活技術,發(fā)現(xiàn)非結構蛋白Nsp1不阻止IRF3的磷酸化。我們利用實時定量PCR的方法,研究表明非結構蛋白Nsp1可以抑制干擾素-p(IFN-p)及熒光素酶的mRNA的轉錄。然后根據(jù)熒光素酶報告分析表明非結構蛋白NSP1對低致病性冠狀病毒的宿主細胞蛋白的合成的影響要強于對SARS-CoV病毒的影響。這些結果表明不同的冠狀病毒可能都可以利用非結構蛋白Nsp1來抵御宿主先天性天然免疫反應和宿主細胞的增殖,然而非結構蛋白Nsp1可能并不是病毒致病性因子中的決定性因子。為了開發(fā)研究針對冠狀病毒的疫苗,我們選擇.了HCoV-229E和HCoV-NL63這兩種人類常見病毒,并利用免疫印跡反應,實時定量PCR和ELISA實驗對它們進行了免疫原性的分析,結果表明HCoV-229E病毒具有極高的免疫原性,并滿足研發(fā)成為病毒疫苗的基本條件。 SARS-CoV出現(xiàn)以后,關于冠狀病毒的抗病毒治療方而的研究越來越多。冠狀病毒Spike刺突糖蛋白的HR2(Heptad Repeat, HR)片段已被證明是有希望用來進行細胞內基因治療的重要靶位點[116,118]。 為了研究HR區(qū)域的免疫原性和中和活性,我們首先對五種已知的冠狀病毒Spike刺突糖蛋白的氨基酸片段進行同源性分析,找出HR2區(qū)域及相鄰上游區(qū)域片段的同源片段序列。接著我們純化出HR2區(qū)域及相鄰上游區(qū)域片段的蛋白,對老鼠進行免疫注射并得到多克隆抗體。通過利用多克隆抗體進行抗體交叉中和活性檢測,我們發(fā)現(xiàn)除了HCoV-HKU1病毒以外,其他四種針對HR2區(qū)域及相鄰上游區(qū)域的抗體均表現(xiàn)出針對同類型病毒或者假病毒的抗體交叉中和活性。接下來我們又進行了針對五種冠狀病毒的Spike刺突蛋白的免疫印跡反應實驗,結果顯示五種人類冠狀病毒中的四種抗HR2的抗體能夠與來自相同或者不同血清群的冠狀病毒的Spike蛋白發(fā)生交叉反應。此外,我們又對豬腸道病毒(TGEV)的HR2區(qū)域及相鄰上游區(qū)域進行了分析,發(fā)現(xiàn)該區(qū)域有著極高的保守性,并對該區(qū)域進行免疫印跡反應實驗和抗體交叉中和活性檢測,結果證明針對TGEV病毒的HR2區(qū)域及相鄰上游區(qū)域的抗體檢測到與HCoV-NL63和HCoV-229E病毒的交叉反應和抗體中和活性。我們的研究結果表明冠狀病毒的HR2區(qū)域及相鄰上游區(qū)域片段是在未來針對已知冠狀病毒或者可能出現(xiàn)新型冠狀病毒在疫苗研發(fā)或者細胞內基因治療方面的重要研究靶位點。 綜上所述,本文中的結果填補了冠狀病毒致病機理研究上的一些空白,促進了關于冠狀病毒疫苗研發(fā)及抗病毒治療方法的研究。
[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.

【學位授予單位】：華東師范大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R373

【共引文獻】

相關期刊論文 前1條

1 趙孟孟;潘俊斌;王衡;;豬流感病毒血凝素基因的研究進展[J];中國畜牧獸醫(yī);2010年11期

相關碩士學位論文 前1條

1 賈園;甲型H1N1流感病毒HA重組蛋白的表達及鑒定[D];吉林農業(yè)大學;2011年

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本文編號：1423743