病毒样蛋白颗粒疫苗胞外自组装机理及过程研究

Virus-like protein particle is formed through static electric and hydrophobic interaction. Because of this weak non-covalent combination, particle’s structure is easily damaged during the chromatographic purification process, resulting in very low recovery and low bioactivity. Moreover, nucleic acid of host cell is often entrained in such particles when assembled in vivo, which will bring potential safety hazard. A new strategy of first purification subunit then making assemby in vitro can overcome these shortcomings, for which proteins self-assembly is the key. Using a fusion of three capsid proteins of enterovirus 71 (EV71) and Hepatitis B virus core antigen (HBc) as models, the sell-assembly mechanism of virus-like protein particles will be investigated in this project. For the case that capsid protein is expressed as inclusions bodies, investigation will focus on uniting the single protein’s refolding with hundreds moleculars nanometer assembly. A number of sophisticated equipment for nanoscale structure analysis, such as asymmetric flow field-flow fractionation, cryo-transmission electron microscopy and atomic force microscopy, will be adopted to reveal the assembling dynamic process, as well as the competition between ordered assembly and non-ordered aggregation. The influences of solution microenvironments on the attraction between inter-units will be studied deeply. New self-assembly methods based on crossflow ultrafiltration and high static pressure will also be established. After finishing this project, a high efficient technique for protein particles self-assembly will be developed, which will not only further improve the modern vaccine preparation platform, but also promote the new kind vaccine’s development.

病毒样蛋白颗粒疫苗由非共价的静电、疏水等弱相互作用结合形成，使得颗粒在纯化及制备过程中容易发生结构破坏或解聚，导致产品收率及活性大幅降低；此外，胞内直接合成的颗粒可能夹带宿主核酸等杂质，致使产品具有安全隐患。先纯化亚基单元后组装成颗粒的制备策略能避免上述问题，其中胞外自组装是关键。本项目以肠道病毒71型三种衣壳蛋白的融合蛋白以及乙肝病毒核心抗原为模型，深入研究病毒样蛋白颗粒胞外自组装机理，发展高效胞外自组装技术。针对常见的包涵体表达，将蛋白质单分子折叠与多分子组装结合在一起，从分子和纳米两个尺度探索颗粒组装机制。采用多种结构表征手段如非对称流场流分级、透射电镜及原子力显微镜等，揭示颗粒组装的动力学途径以及有序组装与无序聚集的竞争机制。深入研究溶液微环境对组装模块间相互作用的影响，建立错流超滤和超高压组装新方法。项目研究结果将完善重组疫苗制备技术平台，推动安全高效的新型疫苗品种发展。

病毒样颗粒（VLP）是高度均一的纳米蛋白颗粒，抗原表位可有序高密度地排列在颗粒表面，能显著增强机体免疫反应。VLP不含病毒核酸，高效安全的独特优势使其在疫苗开发中受到越来越多重视。VLP可通过多种重组体系获得，原核体系制备快速和成本低廉，具有其它体系无法比拟的优势；但也存在目标蛋白可能形成包涵体、完整颗粒会包裹宿主核酸、以及胞内形成的颗粒结构不均一等问题，是原核体系制备VLP的关键技术瓶颈。本项目针对原核体系的这些难点开展研究，取得如下研究成果。.建立两种包涵体的VLP胞外自组装方法。对手足口病毒EV71融合衣壳蛋白包涵体，采用变性纯化、复性、组装分段进行的策略。发现低浓度脲及精氨酸能抑制EV71蛋白复性时形成聚集沉淀，去除脲和精氨酸后的EV71蛋白先组装为五聚体，随后Ca2+诱导下进一步组装形成类病毒颗粒，组装颗粒能诱导诱导小鼠产生高特异性抗体。首次建立SDS/MPD体系的VLP包涵体胞外自组装方法，成功应用于一种基于乙肝病毒核心抗原的嵌合肿瘤疫苗HBc-MAGE3 II包涵体，获得了与HBc天然结构完全一致的HBc-MAGE3 II-VLP。.采用多种手段证实了可溶表达的HBc以及嵌合肿瘤疫苗HBc-MAGE3 I在重组大肠杆菌中存在多种结构，包括VLP、五聚体等组装中间体、以及二聚体的组装单元。提出解聚纯化和纯化后组装策略来提高VLP均一性、纯化效率及核酸去除率。解聚时脲浓度对蛋白收率、纯度、核酸去除率、以及再组装效率有显著影响，尽量降低解聚条件对蛋白二三级结构的破坏。研究HBc-VLP组装动力学过程，发现添加NaCl能提高VLP的组装速率。确定了最优再组装条件，获得不含核酸且形貌完整的HBc-MAGE3 I-VLP。.采用4T1乳腺癌原位模型、转移模型以及预防模型对两种嵌合HBc-VLP开展抗肿瘤活性评价，并与游离抗原表位肽对比确定HBc免疫增强效果。结果证明融合HBc具有明显淋巴结靶向性，HBc-MAGE3 I VLPs能够有效延缓肿瘤生长、抑制肿瘤转移、产生高特异性抗体、促进记忆细胞尤其是效应记忆T细胞分化，还能延长小鼠生存期；与HBc-MAGE3 II VLPs联用后，预防肿瘤效果获得进一步加强，有希望成为新型的预防性肿瘤疫苗。