Title Development of recombinant Yarrowia lipolytica producing virus-like particles of a fish nervous necrosis virus
Author Van-Trinh Luu1, Hye Yun Moon1, Jee Youn Hwang2, Bo-Kyu Kang3, and Hyun Ah Kang1,4*
Address 1Department of Life Science, College of Natural Science, Chung-Ang University, Seoul 06974, Republic of Korea, 2Aquatic Disease Control Division, National Institute of Fisheries Science, Busan 46083, Republic of Korea, 3Green Cross Veterinary Products Co. LTD., Yongin 17066, Republic of Korea, 4Bio-Integration Research Center for Neutra-Pharmaceutical Epigenetics, Chung-Ang University, Seoul 06974, Republic of Korea
Bibliography Journal of Microbiology, 55(8),655–664, 2017,
DOI 10.1007/s12275-017-7218-5
Key Words Yarrowia lipolytica, capsid proteins, multicopy integration, nervous necrosis virus, ribosomal DNA, viruslike particles
Abstract Nervous necrosis virus (NNV) causes viral encephalopathy and retinopathy, a devastating disease of many species of cultured marine fish worldwide. In this study, we used the dimorphic non-pathogenic yeast Yarrowia lipolytica as a host to express the capsid protein of red-spotted grouper nervous necrosis virus (RGNNV-CP) and evaluated its potential as a platform for vaccine production. An initial attempt was made to express the codon-optimized synthetic genes encoding intact and N-terminal truncated forms of RGNNV-CP under the strong constitutive TEF1 promoter using autonomously replicating sequence (ARS)-based vectors. The full-length recombinant capsid proteins expressed in Y. lipolytica were detected not only as monomers and but also as trimers, which is a basic unit for formation of NNV virus-like particles (VLPs). Oral immunization of mice with whole recombinant Y. lipolytica harboring the ARSbased plasmids was shown to efficiently induce the formation of IgG against RGNNV-CP. To increase the number of integrated copies of the RGNNV-CP expression cassette, a set of 26S ribosomal DNA-based multiple integrative vectors was constructed in combination with a series of defective Ylura3 with truncated promoters as selection markers, resulting in integrants harboring up to eight copies of the RGNNVCP cassette. Sucrose gradient centrifugation and transmission electron microscopy of this high-copy integrant were carried out to confirm the expression of RGNNV-CPs as VLPs. This is the first report on efficient expression of viral capsid proteins as VLPs in Y. lipolytica, demonstrating high potential for the Y. lipolytica expression system as a platform for recombinant vaccine production based on VLPs.