Knowledge Management System Of Institute of process engineering,CAS
|Thesis Advisor||苏志国 ; 刘永东|
|Place of Conferral||北京|
|Keyword||肠道病毒71型 多表位融合抗原 手足口病疫苗 两步组合层析 复性及胞外自组装|
手足口病以婴幼儿为易感人群，近年来手足口病的季节性流行严重威胁婴幼儿的健康。肠道病毒71型（EV71）是引起手足口病的主要病原体之一，接种预防疫苗是防控EV71感染的有效手段。不含病毒核酸的病毒样颗粒具有与天然病毒相似的形态结构，集高效性和安全性为一体，是最有开发前景的新型疫苗。目前EV71病毒样颗粒的制备都是用真核表达体系，生产成本高；包裹的宿主核酸和蛋白不易完全去除，因此，存在安全隐患。本论文尝试以成本低廉，规模易放大的E.coli表达体系制备更安全的EV71病毒样颗粒疫苗。首先将EV71病毒衣壳VP1，VP2，VP3上的优势表位抗原片段用两段柔性肽连接起来，EV71衣壳融合抗原蛋白在E.coli中以包涵体形式表达。包涵体溶解后在变性条件下采用Poros HQ的流穿模式和Poros HS的结合模式的组合层析方式纯化，获得纯度高于95%的抗原纯品，过程收率为16.3%。脱除纯化样品中的高浓度脲进行复性，当直接透析至无添加剂的常规缓冲液时蛋白几乎全部沉淀，而先稀释至含2 M脲的pH 8.0的缓冲液然后用G25脱盐柱完全脱除脲则无沉淀形成。透射电镜下观察EV71蛋白形成了约15 nm的粒径均一的五聚体，随后再加入1 mM CaCl2可诱导形成30 nm左右的病毒样颗粒。免疫小鼠能产生高水平的总抗体滴度，体外抗原再刺激能引起脾淋巴细胞增殖，产生高水平的IFN-γ和IL-5，同时激发体液免疫和细胞免疫。本论文首次用原核体系表达含多种结构蛋白的复杂病毒样颗粒，制备的病毒样颗粒为开发新型EV71疫苗奠定了基础，可以为其它类型的病毒样颗粒的原核体系制备提供借鉴。
Human enterovirus 71 (EV71) is one of the main causative pathogens for hand, foot, and mouth disease (HFMD) which often infects infants and children under 5 years old. Prophylactic vaccine is the only way to eradicate the disease of HFMD. Virus-like particles (VLPs), which assemble from viral proteins without infectious nucleic acids, have been shown as promising candidates for EV71 vaccines. However, the EV71 VLPs reported in literature were expressed by eukaryotic cells, which may contain host proteins and DNA, and also cost expensive. Here, a novel EV71 VLP vaccine candidate was developed by self-assembling a fused capsid protein in vitro from inclusion bodies expressed by E.coli.Firstly, a multi-epitope fusion antigen was designed by fusing truncated VP1, VP2 and VP3 of EV71 capsid proteins into one molecule through flexible peptide linkers and expressed in E.coli in the form of inclusion bodies. Then, the solubilized protein was purified at denatured state by two-step ion exchange chromatography with final purity above 95% and a mass recovery of 16.3%. A process of removing the denaturant was necessary for refolding the fused protein. Thus, dialysis was adopted to remove urea in the purified protein. Unfortunately, the protein almost completely precipitated when dialyzing against buffer without any additives. Nevertheless, in the case that the purified protein was firstly diluted in a buffer containing 2 M urea and then exchanged to a buffer (pH 8.0) without urea through a desalting column of G25, no protein precipitate could be found after centrifugation. Transmission electron microscope was applied to observe the morphology of EV71 protein without urea, and uniform pentamers of about 15 nm were found. Moreover, when 1 mM CaCl2 was added, the pantamers self-assembled into 30 nm VLPs. High EV71 specific total IgG titer could be induced by immunization with the assembled VLPs in mice model. Cytokines analysis and IgG subtype detection indicated that fused VLPs induced cellar and also humoral immune responses.Overall， in this paper, a new kind of EV71 VLP vaccine, which could be used for the further pre-clinic assessments, was developed with the advantages of safer and cheaper. And what's more, for the forst time, a complex VLP assembled from three capsid proteins was successfully prepared by protokaryon system.
|薛玲. 肠道病毒71型多表位融合抗原分离纯化及胞外自组装[D]. 北京. 中国科学院研究生院,2016.|
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