Classical swine fever (CSF) is one of the most important viral diseases of swine worldwide. Although live or attenuated virus vaccines have been used to control CSFV, it is difficult to distinguish vaccinated pigs from infected pigs; this leads to restrictions on import and export. Subunit vaccines based on the CSFV E2 glycoprotein have been developed using baculovirus or insect cell systems, but some weaknesses remain. Here, we describe production of an E2 recombinant protein using a Nicotiana benthamiana plant expression system. To do this, we took advantage of the ability of the swine Fc domain to increase solubility and stability of the fusion protein and to strengthen immune responses in target animals. N. benthamiana expressed high amounts of pFc2-fused E2 proteins, which were isolated and purified by affinity chromatography to yield a high pure recombinant protein in a cost-effective manner. Native-polyacrylamide gel electrophoresis and size exclusion chromatography confirmed that the pmE2:pFc2 fusion exists as a multimer rather than as a dimer. Injection of recombinant pmE2 protein into mice or piglets generated anti-pmE2 antibodies with efficient neutralizing activity against CSFV. These results suggest that a purified recombinant E2 protein produced in N. benthamiana generates high titers of neutralizing antibodies in vivo ; as such, the protein could be developed as a subunit vaccine against CSFV.

中文翻译：

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植物中产生的经典猪瘟病毒 E2 融合蛋白在小鼠和猪中引发中和体液免疫反应

经典猪瘟（CSF）是世界范围内最重要的猪病毒性疾病之一。尽管已经使用活疫苗或减毒疫苗来控制 CSFV，但很难区分接种疫苗的猪和受感染的猪；这导致进出口受到限制。已经使用杆状病毒或昆虫细胞系统开发了基于 CSFV E2 糖蛋白的亚单位疫苗，但仍存在一些弱点。在这里，我们描述了使用本氏烟草植物表达系统生产 E2 重组蛋白。为此，我们利用猪 Fc 域的能力来增加融合蛋白的溶解度和稳定性，并加强目标动物的免疫反应。本氏烟草表达大量 pFc2 融合的 E2 蛋白，通过亲和层析分离和纯化，以经济有效的方式产生高纯度的重组蛋白。天然聚丙烯酰胺凝胶电泳和尺寸排阻色谱证实 pmE2:pFc2 融合体以多聚体而不是二聚体形式存在。将重组 pmE2 蛋白注射到小鼠或仔猪体内，产生了对 CSFV 具有有效中和活性的抗 pmE2 抗体。这些结果表明，在本氏烟草中产生的纯化重组 E2 蛋白在体内产生了高滴度的中和抗体。因此，该蛋白质可以开发为针对 CSFV 的亚单位疫苗。将重组 pmE2 蛋白注射到小鼠或仔猪体内，产生了对 CSFV 具有有效中和活性的抗 pmE2 抗体。这些结果表明，在本氏烟草中产生的纯化重组 E2 蛋白在体内产生了高滴度的中和抗体。因此，该蛋白质可以开发为针对 CSFV 的亚单位疫苗。将重组 pmE2 蛋白注射到小鼠或仔猪中，产生了对 CSFV 具有有效中和活性的抗 pmE2 抗体。这些结果表明，在本氏烟草中产生的纯化重组 E2 蛋白在体内产生了高滴度的中和抗体。因此，该蛋白质可以开发为针对 CSFV 的亚单位疫苗。