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. |