Abstract
As one of the most important aquatic fish, Micropterus salmoides suffers lethal and epidemic disease caused by rhabdovirus at the juvenile stage. In this study, a new strain of M. salmoides rhabdovirus (MSRV) was isolated from Yuhang, Zhejiang Province, China, and named MSRV-YH01. The virus infected the grass carp ovary (GCO) cell line and displayed virion particles with atypical bullet shape, 300–500 nm in length and 100–200 nm in diameter under transmission electron microscopy. The complete genome sequence of this isolate was determined to include 11 526 nucleotides and to encode five classical structural proteins. The construction of the phylogenetic tree indicated that this new isolate is clustered into the Vesiculovirus genus and most closely related to the Siniperca chuatsi rhabdovirus. To explore the potential for a vaccine against MSRV, a glycoprotein (1–458 amino acid residues) of MSRV-YH01 was successfully amplified and cloned into the plasmid pFastBac1. The high-purity recombinant bacmid-glycoprotein was obtained from DH10Bac through screening and identification. Based on polymerase chain reaction (PCR), western blot, and immunofluorescence assay, recombinant virus, including the MSRV-YH01 glycoprotein gene, was produced by transfection of SF9 cells using the pFastBac1-gE2, and then repeatedly amplified to express the glycoprotein protein. We anticipate that this recombinant bacmid system could be used to challenge the silkworm and develop a corresponding oral vaccine for fish.
抽象
目 的
探究大口黑鲈弹状病毒株与其他已知弹状病毒的差异, 构建该病毒糖蛋白的杆状病毒表达系统, 用于后续口服疫苗的研发。
创新点
基于获得的大口黑鲈弹状病毒的全基因组序列, 并利用杆状病毒表达系统获得了高丰度的病毒 糖蛋白, 可结合家蚕用于后续口服疫苗的研发。
方 法
利用 cDNA 末端快速扩增法 (RACE) 及逆转录聚合酶链反应 (RT-PCR) 等技术获得了病毒的部分序列, 拼接获得了病毒的全基因组序列。通过 ClustalW 比较该病毒与已知弹状病毒的3′ leader 及 5′ trailer 序列差异, 进一步利用MEGA 软件比较该病毒全基因组序列与其他鱼类弹状 病毒的差异, 确定其系统进化树中的分支及地 位。基于糖蛋白序列分析结果, 利用杆状病毒表 达载体构建该病毒的糖蛋白表达系统, 感染昆虫 卵巢细胞 (SF9) 后, 借助荧光定量 PCR (qRT-PCR)、免疫荧光及蛋白质印迹法 (western blot) 等技术确定病毒增殖及糖蛋白的表达情况。
结 论
该大口黑鲈弹状病毒株全长 11 526 bp, 且与鳜鱼 弹状病毒为同一分支, 属于水泡病毒属。构建的 病毒糖蛋白杆状病毒表达系统成功在 SF9 细胞中 表达了高丰度的糖蛋白。
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Acknowledgments
We thank the Huzhou Fisheries Technical Extension Station (Huzhou, China) for the provision of ill M. salmoides.
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Project supported by the Zhejiang Key R&D Program (No. 2018C02033) and the Zhejiang Provincial Science and Technology Program (No. 2019YSZX0002), China
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Wei-da SHI, Xiao-ying HANG, and Ying-lei WU collected the ill fish. Li LIU designed the project. Sun-jian LYU, Xue-mei YUAN, and Hai-qi ZHANG performed the experiments. Sun-jian LYU performed data analysis and wrote this paper. All authors read and approved the final manuscript. Therefore, all authors have full access to all the data in the study and take responsibility for the integrity and security of the data.
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Sun-jian LYU, Xue-mei YUAN, Hai-qi ZHANG, Wei-da SHI, Xiao-ying HANG, Li LIU, and Ying-lei WU declare that they have no conflict of interest.
All institutional and national guidelines for the care and use of laboratory animals were followed.
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Lyu, Sj., Yuan, Xm., Zhang, Hq. et al. Isolation and characterization of a novel strain (YH01) of Micropterus salmoides rhabdovirus and expression of its glycoprotein by the baculovirus expression system. J. Zhejiang Univ. Sci. B 20, 728–739 (2019). https://doi.org/10.1631/jzus.B1900027
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DOI: https://doi.org/10.1631/jzus.B1900027