Jiangsu Agri-animal Husbandry Vocational College, Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Taizhou, 225300, China.
Jiangsu Agri-animal Husbandry Vocational College, Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Taizhou, 225300, China.
Vet Microbiol. 2018 Feb;215:23-28. doi: 10.1016/j.vetmic.2017.12.020. Epub 2017 Dec 26.
Duck hepatitis A virus (DHAV), a non-enveloped ssRNA virus, can cause a highly contagious disease in young ducklings. The three capsid proteins of VP0, VP1 and VP3 are translated within a single large open reading frame (ORF) and hydrolyzed by protease 3CD. However, little is known on whether the recombinant viral structural proteins (VPs) expressed in insect cells could spontaneously assemble into virus-like particles (VLPs) and whether these VLPs could induce protective immunity in young ducklings. To address these issues, the structural polyprotein precursor gene P1 and the protease gene 3CD were amplified by PCR, and the recombinant proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures and immunogenicity. The recombinant proteins expressed in Sf9 cells were detected by indirect immunofluorescence assay and Western blot analysis. Electron microscopy showed that the recombinant proteins spontaneously assembled into VLPs in insect cells. Western blot analysis of the purified VLPs revealed that the VLPs were composed with the three structural proteins. In addition, vaccination with the VLPs induced high humoral immune response and provided strong protection. Therefore, our findings may provide a framework for development of new vaccines for the prevention of duck viral hepatitis.
鸭甲型肝炎病毒（DHAV）是一种无包膜的 ssRNA 病毒，可导致雏鸭发生高度传染性疾病。衣壳蛋白 VP0、VP1 和 VP3 这三种蛋白由单个大开放阅读框（ORF）翻译，并由蛋白酶 3CD 水解。然而，对于在昆虫细胞中表达的重组病毒结构蛋白（VPs）是否能自发组装成病毒样颗粒（VLPs），以及这些 VLPs 是否能诱导雏鸭产生保护性免疫，目前知之甚少。为了解决这些问题，我们通过 PCR 扩增了结构多蛋白前体基因 P1 和蛋白酶基因 3CD，并使用杆状病毒表达系统在昆虫细胞中表达了重组蛋白，以对其结构和免疫原性进行表征。通过间接免疫荧光和 Western blot 分析检测 Sf9 细胞中表达的重组蛋白。电子显微镜显示，重组蛋白在昆虫细胞中能自发组装成 VLPs。Western blot 分析纯化的 VLPs 表明，VLPs 由三种结构蛋白组成。此外，用 VLPs 进行免疫接种可诱导高体液免疫应答，并提供强有力的保护。因此，我们的研究结果可能为预防鸭病毒性肝炎的新型疫苗的开发提供了框架。
