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Abstract
Nervous necrosis virus (NNV), the etiological agent of viral nervous necrosis, has a high mortality rate of 100% in hatchery-reared larvae and juveniles. At present, there are still no effective vaccines available for NNV. Pichia pastoris surface display of viral capsid proteins was generated in hopes of developing an oral vaccine against red-grouper-nervous-necrosis virus (RGNNV) in fish. Fingerlings or juveniles that showed clinical signs of NNV infection were proved by RT-PCR for the appearance of expected length of 198 bpcDNA and further analysis by DNA sequencing. The DNA fragment containing AGα1 linked to RG-NNVRNA2, 2100 bp in length, was inserted into pPIC9K vector. Linearlized plasmids were electroporated into P. pastoris GS115 (mut+His−) and yeast isolates that had Muts−His+ and resistance phenotype at 4 mg/mL geniticin were selected to determine the integration of the target gene by PCR reaction. The extracted cell walls from the yeasts cultured in buffered-methanol-complex medium (BMMY) through an induction of 0.5% methanol for 6 days, were investigated for the fusion proteins by western blot. A protein band of 73 kDa predicted to be the fusion protein and a non-specific one of 56 kDa were detected. Staining of the fusion proteins expressing cells with corresponding antibodies revealed their presence of NNVRNA2, but varied the intensity of detected signals from cell to cell by confocal laser scanning fluorescence microscopy. The predicted fusion proteins tertiary structure also confirmed exposed conformation of the fusion protein on the cell wall. In this study, the capsid proteins from the red-spotted grouper nervous necrosis virus were successfully expressed on the cell surface of P. pastoris but still low levels of fusion protein expression. Further studies are required to optimize fully surface protein expression prior to evaluate the possible use of the constructed recombinant yeast as an oral vaccine against RG-NNV infection.
Highlights
Aquaculture in Thailand ranks 13th in the world, producing 934.8 million tons of total aquaculture and 19.6 million tons of marine/aquaculture production [1]
This study described the construction of methylotrophic yeast P. pastoris cells displaying RG-Nervous necrosis virus (NNV) capsid protein linked to the N-terminus of the S. cerevisiae GPI-protein, α-agglutinin
RNA2 forms a major open reading frame (ORF) of capsid protein that is responsible for the host-specificity observed among Betanodavirus isolates
Summary
Aquaculture in Thailand ranks 13th in the world, producing 934.8 million tons of total aquaculture and 19.6 million tons of marine/aquaculture production [1]. Oral delivery of vaccines has recently emerged as an attractive alternative to injection in developing countries They enable mass vaccination at a relatively low cost, easy immunization of fish at all stages, and reduce stress from injection site-pain. Proteins displayed on the P. pastoris cell surface were more likely to be correctly folded, and exhibited better stability than that displayed on the S. cerevisiae cell surface [23] It proved to be a good vehicle for delivering antigen to the mucosal epithelium as in bioencapsidated form for larvae fish, and in non-encapsidated form for older fish [24]. Tertiary structure of the fusion protein on the yeast surface was predicted Their immunogenicity and efficacy in protecting fish against RG-NNV by oral delivery might be tested in further studies
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