猪圆环病毒2型(PCV2)是一种全球流行的猪传染性病原体,其衣壳蛋白(Cap)是疫苗开发的唯一关键结构蛋白。先前的研究表明,在大肠杆菌中产生的PCV2Cap蛋白(E.大肠杆菌)可以在体外形成病毒样颗粒(VLP),核定位信号肽(NLS)在稳定PCV2VLP中起关键作用。最近,PCV2d已成为PCV2流行中的重要菌株。在这项研究中,我们系统地优化了PCV2dCap蛋白,并使用大肠杆菌成功地生产了含有NLS的完整PCV2dVLP。通过亲和层析纯化重组PCV2dCap蛋白,每100毫升细菌培养物中产生7.5毫克重组蛋白。我们用精氨酸等各种物质增强了传统的缓冲系统,β-巯基乙醇,甘油,聚乙二醇,和谷胱甘肽促进VLP组装。重组PCV2dCap自组装成直径约20nm的VLP,在优化的缓冲区中具有均匀分布和出色的稳定性。我们开发了疫苗,免疫了猪和小鼠,通过测量PCV2-IgG评估PCV2dVLP疫苗的免疫原性,IL-4,TNF-α,和IFN-γ水平,将它们与利用截短的PCV2Cap抗原的商业疫苗进行比较。HE染色和免疫组织化学测试证实PCV2VLP疫苗提供了强大的保护。结果表明,接种PCV2dVLP疫苗的动物表现出高水平的PCV2抗体,TNF-α和IFN-γ水平在免疫后14天迅速增加,高于市售疫苗中观察到的水平,特别是在小鼠试验中。这可能是由于全长Cap蛋白可以在组装缓冲液中组装成更稳定的PCV2dVLP。总之,与商业疫苗相比,我们生产的PCV2dVLP疫苗在猪和小鼠中引起更强的免疫应答.来自本研究的PCV2dVLPs作为优良的候选疫苗抗原,为PCV2d疫苗研究提供见解。
Porcine circovirus type 2 (PCV2) is a globally prevalent infectious pathogen affecting swine, with its capsid protein (Cap) being the sole structural protein critical for vaccine development. Prior research has demonstrated that PCV2 Cap proteins produced in Escherichia coli (E. coli) can form virus-like particles (VLPs) in vitro, and nuclear localization signal peptides (NLS) play a pivotal role in stabilizing PCV2 VLPs. Recently, PCV2d has emerged as an important strain within the PCV2 epidemic. In this study, we systematically optimized the PCV2d Cap protein and successfully produced intact PCV2d VLPs containing NLS using E. coli. The recombinant PCV2d Cap protein was purified through affinity chromatography, yielding 7.5 mg of recombinant protein per 100 ml of bacterial culture. We augmented the conventional buffer system with various substances such as arginine, β-mercaptoethanol, glycerol, polyethylene glycol, and glutathione to promote VLP assembly. The recombinant PCV2d Cap self-assembled into VLPs approximately 20 nm in diameter, featuring uniform distribution and exceptional stability in the optimized buffer. We developed the vaccine and immunized pigs and mice, evaluating the immunogenicity of the PCV2d VLPs vaccine by measuring PCV2-IgG, IL-4, TNF-α, and IFN-γ levels, comparing them to commercial vaccines utilizing truncated PCV2 Cap antigens. The HE staining and immunohistochemical tests confirmed that the PCV2 VLPs vaccine offered robust protection. The results revealed that animals vaccinated with the PCV2d VLPs vaccine exhibited high levels of PCV2 antibodies, with TNF-α and IFN-γ levels rapidly increasing at 14 days post-immunization, which were higher than those observed in commercially available vaccines, particularly in the mouse trial. This could be due to the fact that full-length Cap proteins can assemble into more stable PCV2d VLPs in the assembling buffer. In conclusion, our produced PCV2d VLPs vaccine elicited stronger immune responses in pigs and mice compared to commercial vaccines. The PCV2d VLPs from this study serve as an excellent candidate vaccine antigen, providing insights for PCV2d vaccine research.