RSV重组蛋白疫苗的制备及其免疫原性和保护性研究

发布时间：2018-07-07 21:24

本文选题：呼吸道合胞病毒(RSV) + 重组融合蛋白　；参考：《中国人民解放军军事医学科学院》2006年博士论文

【摘要】：呼吸道合胞病毒(RSV)是婴幼儿下呼吸道感染的最重要病原，也是造成老年人和免疫缺陷成人高患病率和死亡率的重要原因。60年代研制的福尔马林灭活的RSV(FI-RSV)免疫幼儿后，不仅没达到预防目的，反而使其自然感染RSV后病情加重，，造成80％被免疫者住院，其中2例死亡。目前认为FI-RSV疫苗导致的病情加剧与伴随肺部嗜酸性粒细胞浸润和IL-4、IL-5细胞因子升高的Th2型优势应答、中和抗体水平不足、以及缺乏局部免疫等有关。WHO已将RSV疫苗列为优先发展的疫苗之一，但由于疫苗导致的免疫病理学和安全性问题，目前仍无一例安全有效的疫苗被批准上市。大量研究发现RSV的G蛋白免疫小鼠后能诱导以IgG1为主的抗体和Th2优势应答，但不能诱导IgG2a、Th1和MHC I限制性CD8~+T细胞应答，这种免疫的不平衡性与该类蛋白疫苗的副作用密切相关。研究发现：在诱导CD4~+T细胞的同时激活CD8~+T细胞可以下调Th2型细胞因子，同时防止嗜酸性粒细胞浸涧，可见CD8~+T细胞不仅在清除RSV，而且在下调Th2型细胞因子应答和控制Th2型优势应答导致的病理反应中均起着重要的作用。一个理想的、安全有效的RSV疫苗应该能够同时诱导体液免疫(抗体)、细胞免疫(CTLs)和平衡的Th1／Th2应答。本论文所描述的重组融合蛋白G：125-225-F／M2：81-95(G1F／M2)，包括G蛋白的中和抗体表位片段(G：125-225)和RSV-M2蛋白的CD8~+T细胞表位片段(M2：81-95)；其设计目标是发展一个安全有效的RSV疫苗。首先构建表达质粒并表达纯化融合蛋白G1F／M2：将G1和F／M2：81-95基因片段插入质粒pET-DsbA中构建了原核表达质粒，在E.coli BL21(DE3)中得到表达，采用Ni~+螯合亲和层析法纯化尿素变性的包涵体溶液，梯度透析复性，Western-blot鉴定重组蛋白的RSV特异性，用凝血酶切割DsbA-G1F／M2而得到融合蛋白G1F／M2。免疫原性研究：将融合蛋白G1F／M2以铝盐为佐剂腹腔注射免疫BALB／c小鼠，或将G1F／M2与佐剂型载体热休克蛋白HSP70L1形成的复合物HSP70L1-G1F／M2(HSP-G1F／M2)皮下注射免疫BALB／c小鼠，均诱导了高滴度的蛋白及RSV特异
[Abstract]:Respiratory syncytial virus (RSV) is the most important pathogen of infantile lower respiratory tract infection. It is also an important cause of high morbidity and mortality among the elderly and immunodeficient adults. The formalin inactivated RSV (FI-RSV) developed in the 1960s immunized young children. Not only did not achieve the purpose of prevention, but also made their natural infection with RSV aggravated, resulting in 80% of the immunized patients in hospital, 2 of them died. At present, it is believed that FI-RSV vaccine leads to the exacerbation of the disease and the Th2 predominant response associated with the infiltration of eosinophils and the increase of IL-4 / IL-5 cytokines, but the level of neutralizing antibody is insufficient. Who has listed RSV vaccine as one of the priority vaccines, but due to the immunopathology and safety problems caused by the vaccine, no safe and effective vaccine has been approved to market. A large number of studies showed that RSV G protein immunized mice could induce IgG1 dominant antibody and Th2 dominant response, but could not induce IgG2aTh 1 and MHC I restricted CD8T cell response. The imbalance of this immunity was closely related to the side effects of this kind of protein vaccine. It was found that activation of CD8 ~ T cells at the same time as inducing CD4T cells could down-regulate Th2 cytokines and prevent eosinophilic granulocyte infiltration. CD8T cells play an important role not only in clearing RSVbut also in down-regulating Th2 cytokine response and controlling pathological response induced by Th2 dominant response. An ideal, safe and effective RSV vaccine should be able to induce both humoral (antibody), cellular (CTLs) and balanced Th1 / Th2 responses. The recombinant fusion protein G: 125-225-F / M2: 81-95 (G1F / M2), including G: 125-225 and CD8T epitope of RSV-M2 (M2: 81-95), is designed for the development of a safe and effective RSV vaccine. Firstly, the expression plasmid was constructed and purified fusion protein G1F / M2: 1 and F- / M2: 81-95 were inserted into the plasmid pET-DsbA to construct the prokaryotic expression plasmid, which was expressed in E.coli BL21 (DE3). The urea-denatured inclusion body solution was purified by Ni ~ chelating affinity chromatography. The RSV specificity of the recombinant protein was identified by gradient dialysis renaturation and Western-blot. The fusion protein G1F / M2 was obtained by thrombin cleavage of DsbA-G1F / M2. Immunogenicity study: BALBr / c mice were immunized with the fusion protein G1F / M2 by intraperitoneal injection with aluminum salt as adjuvant, or by subcutaneous injection of the complex HSP70L1-G1F- / M2 (HSP-G1F- / M2), which was formed by G1F / M2 and HSP70L1, respectively. Both of them induced high titer protein and RSV specificity.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392

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