發(fā)布時(shí)間：2018-07-07 21:24

  本文選題：呼吸道合胞病毒(RSV) + 重組融合蛋白�。� 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2006年博士論文

【摘要】：呼吸道合胞病毒(RSV)是嬰幼兒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年代研制的福爾馬林滅活的RSV(FI-RSV)免疫幼兒后，不僅沒(méi)達(dá)到預(yù)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。目前認(rèn)為FI-RSV疫苗導(dǎo)致的病情加劇與伴隨肺部嗜酸性粒細(xì)胞浸潤(rùn)和IL-4、IL-5細(xì)胞因子升高的Th2型優(yōu)勢(shì)應(yīng)答、中和抗體水平不足、以及缺乏局部免疫等有關(guān)。WHO已將RSV疫苗列為優(yōu)先發(fā)展的疫苗之一，但由于疫苗導(dǎo)致的免疫病理學(xué)和安全性問(wèn)題，目前仍無(wú)一例安全有效的疫苗被批準(zhǔn)上市。 大量研究發(fā)現(xiàn)RSV的G蛋白免疫小鼠后能誘導(dǎo)以IgG1為主的抗體和Th2優(yōu)勢(shì)應(yīng)答，但不能誘導(dǎo)IgG2a、Th1和MHC I限制性CD8~+T細(xì)胞應(yīng)答，這種免疫的不平衡性與該類蛋白疫苗的副作用密切相關(guān)。研究發(fā)現(xiàn)：在誘導(dǎo)CD4~+T細(xì)胞的同時(shí)激活CD8~+T細(xì)胞可以下調(diào)Th2型細(xì)胞因子，同時(shí)防止嗜酸性粒細(xì)胞浸澗，可見(jiàn)CD8~+T細(xì)胞不僅在清除RSV，而且在下調(diào)Th2型細(xì)胞因子應(yīng)答和控制Th2型優(yōu)勢(shì)應(yīng)答導(dǎo)致的病理反應(yīng)中均起著重要的作用。 一個(gè)理想的、安全有效的RSV疫苗應(yīng)該能夠同時(shí)誘導(dǎo)體液免疫(抗體)、細(xì)胞免疫(CTLs)和平衡的Th1／Th2應(yīng)答。本論文所描述的重組融合蛋白G：125-225-F／M2：81-95(G1F／M2)，包括G蛋白的中和抗體表位片段(G：125-225)和RSV-M2蛋白的CD8~+T細(xì)胞表位片段(M2：81-95)；其設(shè)計(jì)目標(biāo)是發(fā)展一個(gè)安全有效的RSV疫苗。 首先構(gòu)建表達(dá)質(zhì)粒并表達(dá)純化融合蛋白G1F／M2：將G1和F／M2：81-95基因片段插入質(zhì)粒pET-DsbA中構(gòu)建了原核表達(dá)質(zhì)粒，在E.coli BL21(DE3)中得到表達(dá)，采用Ni~+螯合親和層析法純化尿素變性的包涵體溶液，梯度透析復(fù)性，Western-blot鑒定重組蛋白的RSV特異性，用凝血酶切割DsbA-G1F／M2而得到融合蛋白G1F／M2。 免疫原性研究：將融合蛋白G1F／M2以鋁鹽為佐劑腹腔注射免疫BALB／c小鼠，或?qū)1F／M2與佐劑型載體熱休克蛋白HSP70L1形成的復(fù)合物HSP70L1-G1F／M2(HSP-G1F／M2)皮下注射免疫BALB／c小鼠，均誘導(dǎo)了高滴度的蛋白及RSV特異
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392

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