口蹄疫（Foot-and-mouth disease，FMD）是由FMD病毒（FMDV）引起的一种急性、烈性、高度接触性传染病，主要感染牛、羊、猪等偶蹄类动物。动物感染FMDV后会表现发热症状，并且在口、鼻、舌头或蹄部出现水泡，继而破溃结痂。由于FMD会造成感染动物的生产能力下降，给当地的畜牧养殖业和经济发展造成了严重影响。目前，除了欧洲和美洲的一些发达国家实现了FMD的净化以外，亚洲、部分欧洲、非洲和南美洲等国家和地区的发展中国家仍然有较广泛的流行。FMDV共有A、O、C、Asia1、SAT1、SAT2和SAT3 七个血清型，每种血清型之间没有交叉保护。目前血清C型趋于消失，我国主要流行A型和O型。防治FMD最有效的方法是注射FMDV灭活疫苗。然而，对于FMDV这种烈性传染性病毒，在其疫苗生产过程中还存在诸如需要昂贵的生物隔离施舍和潜在的散毒风险等问题。因此，研发一种更为安全高效的FMDV疫苗尤为重要。

病毒样颗粒（Virus-like particles，VLPs）是由病毒的衣壳蛋白组成，具有类似病毒粒子的形态结构，但不包含病毒核酸，是一种理想的疫苗形式。目前，FMDV VLPs在哺乳动物细胞、昆虫细胞、植物细胞及酿酒酵母等真核细胞中均有成功制备的报道。作为成本较低的真核表达系统，毕赤酵母具有很高的生产VLPs性价比，经济低廉的特点对发展中国家进行生产推广非常友好。然而，有关利用毕赤酵母制备FMDV VLPs的相关研究十分稀少。为此，本研究通过不同的构建策略和寻找最佳的表达方式，同时利用毕赤酵母的翻译后糖基化修饰对FMDV VLPs的理化性质等进行了系统研究，探索利用毕赤酵母表达系统生产FMDV VLPs的可行性，在此基础上对FMDV VLPs的在小鼠和本动物猪进行了免疫原性及效果评估。

1. 采用共表达P1和3C的方式制备FMDV VLPs。在哺乳动物细胞或昆虫细胞中制备FMDV VLPs常采用共表达P1和3C的方式，但是3C通常会造成宿主细胞的死亡。为此，本研究将P1和3C的表达盒插入到同一个pPink-HC载体上来探索该方式在毕赤酵母中的可行性。试验结果显示，P1前体蛋白可以顺利表达但不能被3C切割加工；体外切割试验显示，利用大肠杆菌制备L127P突变的3C蛋白酶可以顺利切割P1，且切割产物可以组装成VLPs；毕赤酵母酸性环境会影响3C蛋白酶的活性。有鉴于此，本研究通过在3C的N端添加可以锚定在核糖体新生肽链出口附近的HLH模式结构，同时对调控3C表达的AOX启动子进行加强性改造以此来提高3C在酵母细胞内对P1的切割效率，最终成功制备了与天然病毒类似的FMDV VLPs，但是切割仍然不完全，存在一定量的中间产物。

2. 采用不依赖3C蛋白酶的方式制备FMDV VLPs。不论3C在哺乳动物细胞的毒性问题还是在毕赤酵母的切割问题，都极大的限制了VLPs的生产和应用。为此，本研究在毕赤酵母中采用同时表达VP0、VP3及VP1蛋白替代3C蛋白酶切割的策略来制备FMDV VLPs。研究结果显示，不同的蛋白在同一个质粒上的表达情况有所不同，VP0的蛋白表达量最高，VP3和VP1的表达量相当，而且目的基因在质粒上的顺序对蛋白的表达也有影响。同时为了便于VLPs的纯化，本试验通过添加His6标签来简化纯化方式。研究结果发现，VP0、VP3及VP1蛋白的N端添加His6标签极大的影响了VLPs的组装。通过对FMDV三级结构进行分析，发现VP3和VP1的C末端因位于VLPs的外部，可以连接亲和标签，VP1的G-H loop环136位插入标签不会影响VLPs组装，为衣壳蛋白的亲和纯化及VLPs组装提供了参考。结果说明通过不依赖3C蛋白酶的方式可成功制备FMDV VLPs，避免了3C蛋白酶在细胞内切割效率不可控的问题及其毒性问题，展示了利用毕赤酵母制备和生产FMDV VLPs的潜力。

3. 制备糖基化修饰的FMDV VLPs。蛋白N-糖基化修饰在蛋白的稳定以及抗原提呈细胞的识别方面具有促进作用。DCs作为功能最强的抗原提呈细胞，其表面存在甘露糖受体（Mannose receptor，MR）可以识别末端为甘露糖的糖链。毕赤酵母作为真核表达系统，可以对蛋白进行翻译后修饰，常被用于研究糖基化修饰对蛋白的影响作用。本研究以FMDV VLPs为模型，通过筛选VP1蛋白潜在的糖基化位点L51、P160和G166来进行糖基化突变，人为引入N-糖基化修饰，成功的利用毕赤酵母表达系统制备出了N-糖基化修饰的VLPs。N-糖谱分析结果显示，携带的聚糖类型多数为高甘露糖型（72.93%），此外还有杂合型（4.16%）和复杂型（22.92%）。VLPs稳定性试验结果显示，糖基化修饰的VLPs比无修饰的VLPs具有更强的稳定性，其在25℃放置72 h后的完整抗原含量仍然保持在60%左右。这些结果说明，通过人为添加糖基化位点可以使蛋白获得糖基化修饰，其对VLPs的组装效率影响不大，但却可以提高VLPs的稳定性及DCs对甘露糖修饰VLPs的提呈作用。

4. FMDV VLPs的免疫原性评估。VLPs具有与病毒粒子类似的形态结构，可刺激机体产生高效全面的免疫反应。本研究利用小鼠和本动物猪验证了毕赤酵母制备的FMDV VLPs的免疫原性。试验结果显示，利用毕赤酵母制备的无糖基化修饰的VLPs免疫受试小鼠14 d后就可检测到高达1:32的特异性抗体水平，直到免疫后42 d仍可检测到高滴度的特异性抗体。糖基化修饰的VLPs对受试小鼠免疫原性的影响存在一定程度的差别，与WT VLPs相比，P160N组产生的特异性抗体和细胞增殖均低于L51T和G166T这两组。免疫后28 d，受试小鼠体内的中和抗体滴度可以达到1:32 ~ 1:64；FMDV VLPs免疫猪的特异性抗体检测结果显示，应用50 μg剂量的VLPs免疫后28 d，WT组和Gly组受试猪血清检测到高达1:22的FMDV中和抗体，而且Gly受试猪的平均中和抗体水平高于WT受试猪；流式细胞和ELISA检测结果显示，WT受试猪和Gly受试猪血清中CD4+和CD8+T淋巴细胞百分比显著高于PBS对照组猪；与WT受试猪相比，Gly受试猪获得更强的细胞免疫反应。免疫后28 d接种同型毒株，WT和Gly受试猪均可达到超过80%保护率，其中Gly受试猪的保护率达到100%。病理学检查结果证实，相较于PBS对照猪，VLPs疫苗免疫猪的脏器组织受到强毒攻击后所遭受的病理性损伤更轻微。上述结果表明，毕赤酵母制备的WT VLPs和糖基化修饰VLPs均可诱导高水平的保护性抗体。

Foot-and-mouth disease (FMD) is an acute, virulent, highly contagious infectious disease caused by the foot-and-mouth disease virus (FMDV), which mainly affects cloven-hoofed animals including cattle, sheep and pigs. Once infected, animals show symptoms of fever and blisters appear on the mouth, nose, tongue, hooves or udder, and these blisters then rupture and form scabs. Infection with FMD leads to a significant reduction in the animal production capacity, which has a negative impact on local livestock production and economic development. At present, although FMD has been eradicated in some developed countries of Europe and America, it is still prevalent in developing countries of Asia, parts of Europe, Africa and South America. FMDV comprises seven serotypes, namely A, O, C, Asia1, SAT1, SAT2 and SAT3, and no cross-protection exists among the serotypes. At present, serotype C is tending to disappear and A and O are the predominant types of FMDV in China. Currently, the most effective way to prevent and control FMD is to inject the inactivated FMD vaccine. However, as a virulent infectious virus, there are some problems in producing inactivated FMD vaccine, such as the need for expensive biocontainment facilities and the potential risk of virus leakage. It is therefore particularly important to develop a safe and effective FMD vaccine.
Virus-like particles (VLPs), which consist of viral capsid proteins and have a morphological structure similar to virions but do not contain viral nucleic acids, are an ideal vaccine form. To date, FMDV VLPs have been successfully produced in mammalian cells, insect cells, plant cells and Saccharomyces cerevisiae. As a low-cost eukaryotic expression system, the production of VLPs using Pichia Pastoris is inexpensive and suitable for production and dissemination in developing countries. Unfortunately, there are few detailed studies on the production of FMDV VLPs using P. pastoris. Therefore，in this study, different construction strategies were adopted, and optimal expression methods were explored. Meanwhile, the effect of glycosylation modification on the physicochemical properties of VLPs was explored by using the post-translational modification function of P. pastoris. On this basis, the immunogenicity and efficacy of FMDV VLPs were evaluated in mice and pigs. 
1. FMDV VLPs were produced by co-expression of P1 and 3C. This approach is commonly used for the production of FMDV VLPs in mammalian or insect cells. However, 3C causes host cell death. In this study, the expression cassettes of P1 and 3C were inserted into the same pPink-HC vector to investigate the feasibility of this method in P. pastoris. The results showed that the P1 precursor protein could be successfully expressed but could not be cleaved by 3C. The L127P mutant 3C protease produced in E. coli was used to cleave P1 in vitro and it could be cleaved successfully and the cleavage products could be assembled into VLPs. The acidic environment of P. pastoris affects the activity of the 3C protease. Thus, an HLH motif structure was added to the N-terminus of 3C, which could be anchored at the exit of the ribosomal nascent peptide chain. Additionally, the AOX promoter that regulates the expression of 3C was strengthened to enhance the cleavage efficiency of 3C on P1. Finally, VLPs similar to those of natural viruses were successfully produced, but there was still some incomplete cleavage and intermediate products remained.
2. 3C protease-independent production of FMDV VLPs. Both the toxicity of 3C in mammalian cells and the cleavage efficiency in P. pastoris severely limit the production and application of VLPs. In this study, the strategy of co-expressing VP0, VP3, and VP1 was adopted in P. pastoris instead of relying on 3C protease cleavage to generate FMDV VLPs.VP0 exhibited the highest protein expression, while VP3 and VP1 were expressed in comparable amounts. The order of the target genes on the plasmid had an effect on the protein expression. To simplify the purification of VLPs, His6 was introduced. However, it was found that His6 added at the N-termini of VP0, VP3, and VP1 significantly hindered VLPs assembly. Through analyzing the tertiary structure of FMDV, it was revealed that the C-termini of VP3 and VP1 are located on the exterior of VLPs, and they can be tagged with affinity tags. Notably, inserting His6 at position 136 of the G-H loop of VP1 does not disrupt VLPs assembly, which offers a reference for the affinity purification and VLPs assembly. In summary, we successfully produced VLPs without 3C protease, circumventing the problems of uncontrollable cleavage efficiency and toxicity of 3C protease in cells. This work also demonstrated the potential of P. pastoris for the production of FMDV VLPs.
3. Preparation of glycosylation-modified FMDV VLPs. N-glycosylation modification plays a facilitating role in protein stabilization and recognition by antigen-presenting cells. DCs, as the most functional antigen-presenting cells, have mannose receptors (MR) on their surface that can recognize glycan chains ending in mannose. P. pastoris, as a eukaryotic expression system, is capable of post-translational modification, and thus is commonly utilized to investigate the impact of glycosylation modification on proteins. In this study, we employed FMDV as a model. By screening potential glycosylation sites L51, P160, and G166 of the VP1 protein for glycosylation mutation, N-glycosylation modification was introduced. Subsequently, N-glycosylated VLPs were successfully produced using P. pastoris. N-glycan profiling revealed that the majority of the carried glycan types were high-mannose type (72.93%). Additionally, there were also hybrid type (4.16%) and complex type (22.92%). The stability assay demonstrated that the glycosylation-modified VLPs exhibited stronger stability compared to the unmodified VLPs. When incubated at 25 °C for 72 h, the intact antigen of the VLPs remained at approximately 60%. These results suggest that the protein can be modified by artificially adding glycosylation sites, which can enhance the stability of VLPs and the presentation of mannose-modified VLPs by DCs, although it has a minimal effect on the assembly efficiency of VLPs.
4. Immunogenicity assessment of FMDV VLPs. VLPs, which mimic the repetitive structures of virions, can induce an efficient and comprehensive immune response. In this study, the immunogenicity of the VLPs produced by P. pastoris was evaluated in mice and pigs. Mice experiments showed that the specific antibodies against FMDV in all experimental groups could be detected at 14 dpi, with titers exceeding 1:32 and that high antibody levels could be maintained until 42 dpi. There were differences in the effects of glycosylation-modified VLPs on immunogenicity, with the P160N group producing lower specific antibodies than the L51T and G166T groups compared to WT VLPs. The neutralizing antibody titers in the mice reached 1:32 - 1:64. The data revealed that the WT and Gly groups exhibited neutralizing antibody titers exceeding 1:22 at 28 dpi, a level that was significantly higher than that observed in the PBS group after immunization with 50 μg VLPs, and the mean neutralizing antibody levels were higher in the Gly group than in the WT group. To evaluate the lymphocyte activation in pigs by VLPs and glycosylation, PBMCs were isolated from immunized pigs at 28 dpi and analyzed by flow cytometry and cytokine levels (IFN-γ, IL-4, TNF-α and IL-1β) were also detected using ELISA kits, and the levels of cytokines in the serum showed that the percentage of CD4+ and CD8+ T-lymphocytes was significantly higher in the WT and Gly groups than the PBS control group. Importantly, the Gly group could stimulate a stronger cellular immune response than the WT group with more than 80% protection after challenge in both the WT and Gly groups and the protection rate of the Gly group reached 100%. Pathological examinations revealed that, compared with the PBS group, the animal organs in the vaccine groups suffered less damage after challenge. These results indicate that both WT VLPs and glycosylated modified VLPs produced by P. pastoris can induce high levels of immune responses.