AiM  Vol.8 No.11 , November 2018
Cell Surface Display of Red-Grouper Nervous Necrosis Virus Capsid Protein on Pichia pastoris
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.
Cite this paper: Intamaso, U. , Chutoam, P. , Poomipak, W. and Pirarat, N. (2018) Cell Surface Display of Red-Grouper Nervous Necrosis Virus Capsid Protein on Pichia pastoris. Advances in Microbiology, 8, 830-845. doi: 10.4236/aim.2018.811055.

[1]   FAO (2016) The State of World Fisheries and Aquaculture 2016 (SOFIA): Contributing to Food Security and Nutrition for All. The State of World Fisheries and Aquaculture, Rome, 204.

[2]   Fukuda, Y.D., Nguyen, H., Furuhashi, M. and Nakai, T. (1996) Mass Mortality of Cultured Sevenband Grouper, Epinephelus septemfasciatus, Associated with Viral Nervous Necrosis. Fish Pathology, 31, 165-170.

[3]   Nishizawa, T., Furuhashi, M., Nagai, T., Nakai, T. and Muroga, K. (1997) Genomic Classification of Fish Nodaviruses by Molecular Phylogenetic Analysis of the Coat protein Gene. Applied and Environmental Microbiology, 63, 1633-1636.

[4]   Johansen, R., Grove, S., Svendsen, A.K., Modahl, I. and Dannevig, B. (2004) A Sequential Study of Pathological Findings in Atlantic Halibut, Hippoglossus hippoglossus (L.), throughout One Year after an Acute Outbreak of Viral Encephalopathy and Retinopathy. Journal of Fish Diseases, 27, 327-341.

[5]   Moody, N.J.G, Horwood, P.F., Reynolds, A., Mahony, T.J., Anderson, I.G. and Oakey, H.J. (2009) Phylogenetic Analysis of Betanodavirus Isolates from Australian Finfish. Diseases of Aquatic Organisms, 87, 151-160.

[6]   Nakai, T., Mori, K., Sugaya, T., Nishioka, T., Mushiake, K. and Yamashita, H. (2009) Current Knowledge on Viral Nervous Necrosis (VNN) and Its Causative Betanodaviruses. Israeli Journal of Aquaculture—Bamidgeh, 61, 198-207.

[7]   Boonyaratpalin, S., Supamattaya, K., Kasornchandra, J. and Hoffmann, R.W. (1996) Picorna-Like Virus Associated with Mortality and a Spongious Encephalopathy in Grouper Epinephelus malabaricus. Diseases of Aquatic Organisms, 26, 75-80.

[8]   Danayadol, Y. (1995) Viral Nervous Necrosis in Brownspotted Grouper, Epinephelus malabaricus, Cultured in Thailand. Diseases in Asian Aquaculture, 2, 227-233.

[9]   Roonngkamnertwongsa, S., Kanchanakhan, S., Danayadol, Y. and Direkbusarakom, S. (2005) Identification of Betanodavirus Isolated from Viral Necrosis Disease in Red-Spotted Grouper, Epinephilus coides Cultured in Thailand Using PCR and Sequence Analysis. In: Walker, P., Lester, R. and Bondad-Reantaso, M.G., Eds., Disease in Asian Aquaculture, Fish Health Section, Asian Fisheries Society, Manila, 207-216.

[10]   Keawcharoen, J., Techangamsuwan, S., Ponpornpisit, A., Lombardini, E.D., Patchimasiri, T. and Pirarat, N. (2015) Genetic Characterization of a Betanodavirus Isolated from a Clinical Disease Outbreak in Farm-Raised Tilapia Oreochromis niloticus (L.) in Thailand. Journal of Fish Diseases, 38, 49-54.

[11]   Munday, B.L., Kwang, J. and Moody, N. (2002) Betanodavirus Infections of Teleost Fish: A Review. Journal of Fish Diseases, 25, 127-142.

[12]   Comps, M., Pepin, J.F. and Bonami, J.R. (1994) Purification and Characterization of Two Fish Encephalitis Viruses (FEV) Infecting Lates calcarifer and Dicentrarchus labrax. Aquaculture (Netherlands), 123, 1-10.

[13]   Mori, K., Mushiake, K. and Arimoto, M. (1998) Control Measures for Viral Nervous Necrosis in STRIPED jack [Pseudocaranx dentex]. Fish Pathology (Japan), 33, 443-444.

[14]   Mushiake, K., Nishizawa, T., Nakai, T., Furusawa, I. and Muroga, K. (1994) Control of VNN in Striped Jack: Selection of Spawners Based on the Detection of SJNNV Gene by Polymerase Chain Reaction (PCR). Fish Pathology, 29, 177-182.

[15]   Watanabe, K.I., Nishizawa, T. and Yoshimizu, M. (2000) Selection of Brood Stock Candidates of Barfin Flounder Using an ELISA System with Recombinant Protein of Barfin Flounder Nervous Necrosis Virus. Diseases of Aquatic Organisms, 41, 219-223.

[16]   Czerkinsky, C. and Holmgren, J. (2009) Enteric Vaccines for the Developing World: A Challenge for Mucosal Immunology. Mucosal Immunology, 2, 284-287.

[17]   Ananphongmanee, V., Srisala, J., Sritunyalucksana, K. and Boonchird, C. (2015) Yeast Surface Display of Two Proteins Previously Shown to Be Protective against White Spot Syndrome Virus (WSSV) in Shrimp. PLoS ONE, 10, e0128764.

[18]   Park, M., Suh, S.-S., Hwang, J., Kim, D., Jongbum, P. and Chung, Y-J. (2014) Production of Red-Spotted Grouper Nervous Necrosis Virus (RGNNV) Capsid Protein Using Saccharomyces cerevisiae Surface Display. Journal of Life Science, 24, 995-1000.

[19]   Kondo, A. and Ueda, M. (2004) Yeast Cell-Surface Display—Applications of Molecular Display. Applied Microbiology and Biotechnology, 64, 28-40.

[20]   Sakuragi, H., Kuroda, K. and Ueda, M. (2011) Molecular Breeding of Advanced Microorganisms for Biofuel Production. Journal of Biomedicine and Biotechnology, 2011, Article ID: 416931.

[21]   Tanaka, T., Yamada, R., Ogino, C. and Kondo, A. (2012) Recent Developments in Yeast Cell Surface Display toward Extended Applications in Biotechnology. Applied Microbiology and Biotechnology, 95, 577-591.

[22]   Cregg, J.M., Cereghino, J.L., Shi, J. and Higgins, D.R. (2000) Recombinant Protein Expression in Pichia pastoris. Molecular Biotechnology, 16, 23-52.

[23]   Jiang, Z., Gao, B., Ren, R., Tao, X., Ma, Y. and Wei, D. (2008) Efficient Display of Active Lipase LipB52 with a Pichia pastoris Cell Surface Display System and Comparison with the LipB52 Displayed on Saccharomyces cerevisiae Cell Surface. BMC Biotechnology, 8, 4.

[24]   Embregts, C.W.E., Reyes-Lopez, F., Pall, A.C., Stratmann, A., Tort, L. and Lorenzen, N. (2018) Pichia pastoris Yeast as a Vehicle for Oral Vaccination of Larval and Adult Teleosts. Fish & Shellfish Immunology.

[25]   Nishizawa, T., Mori, K., Furuhashi, M., Nakai, T., Furusawa, I. and Muroga, K. (1995) Comparison of the Coat Protein Genes of Five Fish Nodaviruses, the Causative Agents of Viral Nervous Necrosis in Marine Fish. The Journal of General Virology, 76, 1563-1569.

[26]   Wasilenko, J.L., Sarmento, L., Spatz, S. and Pantin-Jackwood, M. (2010) Cell Surface Display of Highly Pathogenic Avian Influenza Virus Hemagglutinin on the Surface of Pichia pastoris Cells Using Alpha-Agglutinin for Production of Oral Vaccines. Biotechnology Progress, 26, 542-547.

[27]   Roy, A., Kucukural, A. and Zhang, Y. (2010) I-TASSER: A Unified Platform for Automated Protein Structure and Function Prediction. Nature Protocols, 5, 725-738.

[28]   Yang, J., Yan, R., Roy, A., Xu, D., Poisson, J. and Zhang, Y. (2015) The I-TASSER Suite: Protein Structure and Function Prediction. Nature Methods, 12, 7-8.

[29]   Zhang, Y. (2008) I-TASSER Server for Protein 3D Structure Prediction. BMC Bioinformatics, 9, 40.

[30]   Cregg, J.M. and Madden, K.R. (1987) Development of Yeast Transformation Systems and Construction of Methanol-Utilization-Defective Mutants of Pichia pastori by Gene Disruption.

[31]   Chiruvolu, V., Cregg, J.M. and Meagher, M.M. (1997) Recombinant Protein Production in an Alcohol Oxidase-Defective Strain of Pichia pastoris in Fedbatch Fermentations. Enzyme and Microbial Technology, 21, 277-283.

[32]   Tschopp, J.F., Sverlow, G., Kosson, R., Craig, W. and Grinna, L. (1987) High-Level Secretion of Glycosylated Invertase in the Methylotrophic Yeast, Pichia pastoris. Nature Biotechnology, 5, 1305-1308.

[33]   Scorer, C.A., Clare, J.J., McCombie, W.R., Romanos, M.A. and Sreekrishna, K. (1994) Rapid Selection Using G418 of High Copy Number Transformants of Pichia pastoris for High-Level Foreign Gene Expression. Nature Biotechnology, 12, 181-184.

[34]   Clare, J.J., Romanes, M.A., Rayment, F.B., Rowedder, J.E., Smith, M.A. and Payne, M.M. (1991) Production of Mouse Epidermal Growth Factor in Yeast: High-Level Secretion Using Pichia pastoris Strains Containing Multiple Gene Copies. Gene, 105, 205-212.

[35]   Brake, A.J., Merryweather, J.P., Coit, D.G., Heberlein, U.A., Masiarz, F.R. and Mullenbach, G.T. (1984) Alpha-Factor-Directed Synthesis and Secretion of Mature Foreign Proteins in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences of the United States of America, 81, 4642-4646.

[36]   Chen, N.-C., Yoshimura, M., Guan, H.-H., Wang, T.-Y., Misumi, Y. and Lin, C.-C. (2015) Crystal Structures of a Piscine Betanodavirus: Mechanisms of Capsid Assembly and Viral Infection. PLoS Pathogens, 11, e1005203.

[37]   Costa, J.Z. and Thompson, K.D. (2016) Understanding the Interaction between Betanodavirus and Its Host for the Development of Prophylactic Measures for Viral Encephalopathy and Retinopathy. Fish & Shellfish Immunology, 53, 35-49.

[38]   Schreuder, M.P., Mooren, A.T., Toschka, H.Y., Verrips, C.T. and Klis, F.M. (1996) Immobilizing Proteins on the Surface of Yeast Cells. Trends in Biotechnology, 14, 115-120.

[39]   Luu, V.-T., Moon, H.Y., Hwang, J.Y., Kang, B.-K. and Kang, H.A. (2017) Development of Recombinant Yarrowia lipolytica Producing Virus-Like Particles of a Fish Nervous Necrosis Virus. Journal of Microbiology, 55, 655-664.