新城疫病毒样颗粒的构建及免疫原性和免疫机制研究

发布时间：2018-01-01 07:33

本文关键词：新城疫病毒样颗粒的构建及免疫原性和免疫机制研究　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：病毒样颗粒(Virus-like particles,VLPs)是由病毒结构蛋白装配而成的空心蛋白颗粒,与真实病毒形态结构相似,无自主复制能力,可作为安全有效的新型疫苗预防病毒性传染病,近年来VLP技术已成为疫苗研究领域的热点。新城疫病毒样颗粒(Newcastle disease virus-like particles,NDV VLPs)是由新城疫病毒基质蛋白(M)为病毒骨架,可装配病毒血凝素-神经氨酸酶(HN)、融合蛋白(F)或核蛋白(NP)。已有研究表明,NDV VLPs能诱导机体产生特异性体液和细胞免疫应答,以及有效的免疫保护效力,这与其他VLP疫苗诱导的适应性免疫应答能力相似。然而,关于NDV VLPs如何激活天然免疫应答的研究尚无报道。正常情况下,机体免疫系统识别外源抗原涉及特定的免疫细胞,如吞噬细胞或淋巴细胞。树突状细胞(Dendritic cells,DCs)是目前抗原递呈能力最强的专职免疫细胞,在连接天然免疫与适应性免疫之间具有独特功能。成熟DCs(m DCs)迁移至次级淋巴组织能够刺激初始型T细胞(Na?ve T cell)活化和增殖,被认为是特异性免疫应答的始动者。因此,阐明NDV VLPs诱导DC成熟与迁移的免疫机制对更好地理解VLP激活的天然免疫应答具有重要科学意义。目前,有关DC成熟与迁移的研究主要集中在小鼠等哺乳动物模型上,虽然NDV感染的宿主为禽类,但是已经明确禽类的免疫反应大部分与其他生物物种相同,只是在细节上存在差异。为此,本研究以NDV VLPs与小鼠DCs为研究靶点,利用昆虫杆状病毒表达系统组装了NDV VLPs,通过小鼠模型分析NDV VLPs激发的体液和细胞免疫应答水平以评价其免疫原性;利用分子生物学方法、细胞生物学分析手段以及系统免疫学技术体系,探讨NDV VLPs诱导体外DC成熟与迁移的分子机制及其信号通路,揭示体内DCs捕获NDV VLPs后的迁移路径及递呈效应。主要研究内容如下:一、本实验首先利用昆虫杆状病毒表达系统,构建共表达NDV结构蛋白的重组杆状病毒(r BV-M和r BV-M+HN),组装并纯化了NDV VLPs(M和M-HN)。免疫电镜检测结果可见明显胶体金颗粒,表明NDV VLPs能与NDV HN蛋白单克隆抗体发生特异性反应,同时可见病毒颗粒具有典型的囊膜结构,与野生型NDV大小相似(100-200nm)。Western blot分析结果可见约40k Da和68k Da大小条带,表明该颗粒还能与特异性NDV抗体发生反应。为评价NDV VLPs在小鼠模型中的免疫原性,血凝抑制试验和间接ELISA法检测结果表明VLP组可诱导产生更高的血凝抑制抗体效价和特异性Ig G抗体水平,与异源病毒(La Sota株)的反应原性一致,同时也发现VLP组Ig G抗体亚型以Ig G1为主。流式细胞术检测结果表明,VLPs可诱导产生较多的Th细胞(CD3+CD4+细胞),并且相比于WIV组能激活更多的Th1细胞(CD4+IFN-γ+细胞)和Tc细胞(CD8+IFN-γ+细胞);另外还发现VLPs招募了更多的DC迁移至脾脏。以上结果表明,NDV VLPs能产生特异性免疫应答,这可能与招募的DC活化程度有关。二、为探索NDV VLPs诱导体外DC成熟与迁移的免疫机制,采用体外重组细胞因子GM-CSF和IL-4联合诱导小鼠骨髓细胞分化为具有典型树枝状突起DCs,且表面标志分子CD11c和MHC II符合后续实验的要求。以原代细胞开展系统性DC免疫学研究,FITC-葡聚糖吞噬实验结果表明DCs可有效摄取VLPs;混合淋巴细胞反应数据说明,VLP组DCs可促进初始型T细胞分化,具体表现为Th1型(IL-2)和Th2型(IL-10)细胞因子分泌水平增加;流式细胞术检测表面成熟标志分子和ELISA法检测细胞因子结果可见,VLPs能诱导DC上调MHC II、CD80、CD86、CD40分子的表达以及增加TNF-α、IFN-γ、IL-6、IL12p70的分泌;另外,还发现单独M VLPs或是HN蛋白也能促进炎性细胞因子的分泌,这表明完整的病毒粒子结构和HN蛋白也可诱导DC成熟;然而不同组装类型的VLPs其分泌炎性细胞因子的水平不一,推测这可能与NDV F或HN特性有关。为进一步探讨NDV VLPs诱导DC成熟的免疫机制及其可能的信号通路,流式细胞术和ELISA法检测结果表明,TLR4和NF-κB抑制剂处理能降低NDV VLPs诱导的DC活化,表现为DC表面成熟标志分子表达不同程度降低并且分泌细胞因子水平显著减少;Western blot结果显示NDV VLPs促进了DC胞内NF-κB(p65)、IκB以及IKK分子的磷酸化表达,说明VLPs诱导DC成熟可通过TLR4-IKK-IκB-NF-κB信号通路实现。与此同时,VLPs可上调DC表面CCR7分子的表达,而下调CCR5分子的表达;进一步采用Transwell小室法和抗体阻断法验证了VLPs诱导DC迁移依赖于CCR7-CCL19/CCL21轴的作用,并且CCL21的作用更为显著。三、为了揭示NDV VLPs诱导体内DC成熟与迁移的细胞与分子机制,本实验先采用CFSE标记m DCs经尾静脉过继回输给小鼠,流式细胞术检测引流淋巴结和脾脏并结合ELISA法测定趋化因子结果表明,m DCs在体内的迁移仍是依赖CCL19和CCL21的浓度差作用,负载VLPs的DCs可促进淋巴组织中CCL19和CCL21的分泌,并且CCL21对m DC迁移更为重要;最终通过流式细胞术检测细胞表型以及胞内细胞因子结果显示m DC组各亚型T细胞显著高于im DC组和PBS组,表明该m DCs能够在体内激活脾脏T细胞并向各亚群分化。为了模拟真实VLPs在体内被处理加工的过程,本实验另外也注射VLPs至小鼠足底,结果表明VLPs早期招募DCs在引流淋巴结聚集,而随着时间推移,脾脏DCs的百分比上升,推测DC是由外周组织经引流淋巴结向脾脏迁移,而72h后脾脏T、B淋巴细胞活化和增殖导致DC百分占比率降低。另外,胞内细胞因子染色结果表明仅Th型(CD4+IFN-γ+和CD4+IL-4+)细胞产生了分化,说明VLPs被体内DCs摄取进而激活初始型T细胞。以上结果显示,VLPs能诱导体内DC经由引流淋巴结迁移至脾脏,进而促使初始型T细胞活化和分化。综上所述,本研究组装了NDV VLPs,该颗粒具有良好的免疫原性;揭示NDV VLPs诱导体内、外DC成熟与迁移的免疫机制,为深入阐明VLPs激活天然免疫应答机制提供科学依据。
[Abstract]:Virus like particles (Virus-like, particles, VLPs) is hollow particles by protein structure protein of virus assembly, similar to the real virus morphology, no self replication ability, can be used as a safe and effective vaccine to prevent viral infectious disease in recent years, VLP technology has become a hot research field for vaccine of Newcastle disease virus like. The particles (Newcastle disease virus-like particles, NDV VLPs) by Newcastle disease virus matrix protein (M) virus skeleton assembly virus hemagglutinin neuraminidase (HN), fusion protein (F) or nuclear protein (NP). Studies have shown that NDV VLPs can induce specific humoral and cellular immune responses. And the effective immune protection effect, which induced by VLP vaccine and other adaptive immune response is similar. However, there are few research reports about NDV VLPs how to activate the innate immune response. Under normal circumstances, machine Specific immune cells involved in the immune system to recognize the foreign body antigen, such as macrophages or lymphocytes. Dendritic cells (Dendritic cells DCs) is currently a full-time antigen presenting immune cells has the strongest ability, in the connection between innate immunity and adaptive immunity unique function. The mature DCs (m DCs) can stimulate migration to secondary lymphoid tissue the initial T cell (Na ve? T cell) activation and proliferation, is considered to be the initiator of specific immune response. Therefore, clarifying the mechanism of NDV VLPs immune maturation and migration induced by DC has important scientific significance for a better understanding of the innate immune response activated by VLP. At present, the research on DC maturation and migration of the main in mice and other mammals model, although the NDV infected host for poultry, but has a clear immune response of poultry and other biological species, most of the same, but the details are poor Different. Therefore, in this study, NDV VLPs and DCs mice as the research target, using insect baculovirus expression system was assembled NDV VLPs, to evaluate the immunogenicity of NDV VLPs cells and stimulate the humoral immune response level by mouse model analysis; with the method of molecular biology, cell biology and Immunology technology system system analysis method to explore the molecular mechanism of NDV and VLPs in vitro maturation and migration induced by DC and its signal transduction pathway, migration path to capture NDV after VLPs and DCs revealed in presenting effect. The main research contents are as follows: first, this study first used the insect baculovirus expression system, recombinant baculovirus coexpressing NDV structural proteins (R and BV-M R BV-M+HN), the assembly and the purification of NDV (M VLPs and M-HN). Immune electron microscopy results showed that the colloidal gold particles, NDV VLPs and NDV HN protein monoclonal antibody specific against At the same time, visible viral particles with envelope structure typical, similar to wild type NDV (100-200nm).Western size blot analysis results about 40K Da and 68K Da visible stripe, but also shows that the particles with specific NDV antibody reaction. For evaluation of NDV VLPs in a mouse model of immunogenicity, blood coagulation inhibition test and indirect ELISA assay results show that VLP can induce higher hemagglutination inhibition antibody titer and specificity of Ig G antibody level, with heterologous virus (La strain Sota) the reactionogenicity, also found that group VLP Ig G to Ig G1 antibody subtype. Flow cytometry the results show that VLPs can induce more Th cells (CD3+CD4+ cells), and compared to the WIV group can activate more Th1 cells (CD4+IFN- + cells) and Tc cells (CD8+IFN- + cells); it is also found that VLPs recruited more DC migrate to the node above the spleen. The results show that NDV VLPs can produce specific immune response, which may be related to the recruitment of DC activation degree. Two, to explore the mechanism of NDV VLPs DC in vitro immune maturation and migration induced by in vitro, recombinant cytokines GM-CSF and IL-4 combination induced differentiation of bone marrow cells in mice with typical dendritic processes of DCs, and the surface markers CD11c and MHC II in accordance with the requirements of the following experiment. To carry out the research of DC system based on immunology primary cells, FITC- glucan phagocytosis experiment results show that DCs can effectively absorb VLPs data; mixed lymphocyte reaction, VLP group of DCs may promote the differentiation of naive T cells, the specific performance of Th1 type (IL-2) and Th2 (IL-10) the secretion of cytokines were increased; the detection of surface markers of molecules and ELISA cytokines were measured by flow cytometry showed that VLPs could induce the upregulation of MHC, DC II, CD80, CD86, CD40 and increase the expression of the son TNF- alpha, IFN- gamma, IL-6, IL12p70 secretion; in addition, also found that M alone VLPs or HN protein can promote the secretion of inflammatory cytokines, suggesting that the virus particle structure and complete HN protein can also induce DC maturation; however, different types of VLPs assembled the secretion of inflammatory cytokines no, NDV and F speculate that this may or HN characteristics. To further explore the NDV signaling pathway in VLPs induced immune mechanism of DC maturation and the possibility of the flow cytometry and ELISA assay showed that TLR4 and NF- kappa B inhibitor NDV treatment could reduce VLPs induced DC activation, as mature DC surface molecules different expression levels decreased and cytokine secretion was significantly reduced Western blot signs; results showed that NDV VLPs promoted DC intracellular NF- kappa B (p65), the expression of I kappa B and IKK molecules. The phosphorylation of VLPs induced by B-NF- DC mature TLR4-IKK-I kappa kappa B The signal pathway of VLPs. At the same time, DC can up regulate the expression of CCR7 molecules on the surface, and the expression of CCR5 molecules; further using Transwell assay and antibody blocking method to verify the VLPs induced DC migration depends on the role of CCR7-CCL19/CCL21 axis, and the effect of CCL21 is more significant. Three, in order to reveal the cellular and molecular mechanisms of NDV VLPs in vivo maturation and migration induced by DC, the first experiment using CFSE labeled m DCs via intravenous adoptive transfer to mice, flow cytometry in draining lymph nodes and spleen combined with ELISA method for the determination of chemokines results showed that the concentration of M is still in the migration of DCs in CCL19 dependent and CCL21 difference, VLPs load the DCs can promote the secretion of CCL19 and CCL21 in lymphoid tissues, and CCL21 of the m DC migration is more important; finally through flow cytometry to detect the phenotype and intracellular cytokines showed m DC in each group Subtype of T cells was significantly higher than that of im DC group and PBS group, indicating that the m DCs can be activated in vivo spleen T cell subsets and differentiation. In order to simulate the process of VLPs in vivo was processed, this experiment also injected VLPs mice to the foot, the results show that the VLPs early recruitment of DCs gathered in the draining lymph node however, with the passage of time, the percentage of DCs in spleen increased, suggesting that DC is a peripheral tissue by draining lymph nodes to the spleen and spleen after migration, 72h T, B lymphocyte activation and proliferation in DC percent ratio decreased. In addition, intracellular cytokine staining results showed that only Th (CD4+IFN- + and CD4+IL-4+) cells produced a differentiation, indicating that VLPs is in DCs uptake and activation of naive T cells. These results showed that VLPs could induce DC in vivo via lymph nodes migrate to the spleen, which prompted the initial T cell activation and differentiation. To sum up, this research The NDV VLPs was assembled. The particle has good immunogenicity. It reveals the immune mechanism of NDV VLPs inducing the maturation and migration of DC in vivo and in vitro, and provides a scientific basis for further elucidating the mechanism of VLPs activating the innate immune response.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.4

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