Porcine circovirus type 2 (PCV2) is an economically important pathogen affecting swine industry worldwide. The production of current PCV2 vaccines is time-consuming and expensive. Elastin-like polypeptides (ELP) undergo temperature-dependent inverse phase transition and ELPylated proteins can be purified simply by inverse transition cycling (ITC). The Cap protein of PCV2b, together with the virus neutralizing (VN) epitopes of PCV2a, PCV2d and PCV2e, was expressed in E. coli as an ELPylated protein, and purified by ITC in the presence of mild detergents. For the control purpose, the Cap protein was also expressed as a His-tagged protein and purified by nickel affinity chromatography. The formation of ELPylated VLP (ELP-VLP) and His-tagged VLP (VLP) was revealed by transmission electron microscopy. Mice were immunized two times with the two forms of VLP and the antigen-specific IgG antibody, VN antibody, cytokine responses and immunoprotection against PCV2 challenge were compared. ELPylated Cap protein was expressed as a soluble protein and purified to 94.3% purity by ITC in the presence of 1% Triton X-100 and 0.5 M urea. His-tagged Cap fusion protein was expressed as insoluble inclusion bodies and purified to 90% purity under denatured conditions. The two purified fusion proteins assembled into VLP with similar morphology. Compared to immunization with VLP, immunization with ELP-VLP induced significantly (p < 0.01) stronger VN antibody response and slightly (p < 0.05) stronger Cap-specific IgG antibody response, cytokine production and immunoprotection against PCV2 challenge. A novel ELPylation platform for easy preparation of PCV2 VLP was established and the prepared ELP-VLP was more immunogenic than VLP. The ELPylation technology could be used for other VLP preparation and the prepared ELP-VLP could be developed as a novel PCV2 subunit vaccine.

中文翻译：

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猪圆环病毒2型ELP化病毒样颗粒的产生和免疫原性评估

猪圆环病毒2型（PCV2）是一种经济重要的病原体，会影响全世界的养猪业。当前的PCV2疫苗的生产既费时又昂贵。弹性蛋白样多肽（ELP）经历温度依赖性的逆相转变，并且可以通过逆转变循环（ITC）简单地纯化ELPylated蛋白。PCV2b的Cap蛋白与PCV2a，PCV2d和PCV2e的病毒中和（VN）表位一起在大肠杆菌中表达为ELPylated蛋白，并在温和的去污剂存在下通过ITC纯化。出于对照目的，Cap蛋白也表达为His-标记蛋白，并通过镍亲和色谱法纯化。通过透射电子显微镜揭示了ELP化的VLP（ELP-VLP）和His标记的VLP（VLP）的形成。用两种形式的VLP免疫小鼠两次，并比较了抗原特异性IgG抗体，VN抗体，细胞因子应答和针对PCV2攻击的免疫保护作用。ELPylated Cap蛋白表达为可溶性蛋白，并在1％Triton X-100和0.5 M尿素存在下通过ITC纯化至94.3％的纯度。His标记的Cap融合蛋白表达为不溶性包涵体，并在变性条件下纯化至90％的纯度。两种纯化的融合蛋白组装成具有相似形态的VLP。与用VLP免疫相比，用ELP-VLP免疫可显着（p <0.01）增强VN抗体反应，而对Cap特异性IgG抗体，细胞因子产生和针对PCV2攻击的免疫保护则稍强（p <0.05）。建立了易于制备PCV2 VLP的新型ELPylation平台，制备的ELP-VLP比VLP具有更高的免疫原性。ELPylation技术可用于其他VLP制备，制备的ELP-VLP可开发为新型PCV2亚基疫苗。