Journal of Microbiology

Journal Article

Development of recombinant Yarrowia lipolytica producing virus-like particles of a fish nervous necrosis virus: Van-Trinh Luu , Hye Yun Moon , Jee Youn Hwang , Bo-Kyu Kang , Hyun Ah Kang; J. Microbiol. 2017;55(8):655-664. Published online July 28, 2017; DOI: https://doi.org/10.1007/s12275-017-7218-5

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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.

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Elucidation and engineering of Sphingolipid biosynthesis pathway in Yarrowia lipolytica for enhanced production of human-type sphingoid bases and glucosylceramides
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Metabolic Engineering.2025; 87: 68. CrossRef
Yeast-Based Virus-like Particles as an Emerging Platform for Vaccine Development and Delivery
Vartika Srivastava, Kripa N. Nand, Aijaz Ahmad, Ravinder Kumar
Vaccines.2023; 11(2): 479. CrossRef
Humoral immune response in Asian seabass vaccinated with inactivated and recombinant viral nervous necrosis vaccine
M. Makesh, N. Venkata Satyanarayana, K. Muddukrishnaiah, Sujeet Kumar, G. Thiagarajan, Ashok Kumar Jangam, R. Subburaj, M. Kailasam, K.K. Vijayan
Aquaculture.2023; 569: 739384. CrossRef
Biomanufacturing of γ-linolenic acid-enriched galactosyldiacylglycerols: Challenges in microalgae and potential in oleaginous yeasts
Xiaosong Gu, Lei Huang, Jiazhang Lian
Synthetic and Systems Biotechnology.2023; 8(3): 469. CrossRef
Yeast as carrier for drug delivery and vaccine construction
Yifu Tan, Liwei Chen, Ke Li, Beibei Lou, Yanfei Liu, Zhenbao Liu
Journal of Controlled Release.2022; 346: 358. CrossRef
Construction of Attenuated Strains for Red-Spotted Grouper Nervous Necrosis Virus (RGNNV) via Reverse Genetic System
Yingying Lei, Yu Xiong, Dagang Tao, Tao Wang, Tianlun Chen, Xufei Du, Gang Cao, Jiagang Tu, Jinxia Dai
Viruses.2022; 14(8): 1737. CrossRef
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Hang Su, Igor A. Yakovlev, André van Eerde, Jianguo Su, Jihong Liu Clarke
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Ting Gao, Caixia Gao, Siyu Wu, Yingying Wang, Jiyuan Yin, Yingying Li, Weiwei Zeng, Sven M. Bergmann, Qing Wang
Vaccines.2021; 9(1): 53. CrossRef
Developing oral nanovaccines for fish: a modern trend to fight infectious diseases
Carlos Angulo, Marlene Tello‐Olea, Martha Reyes‐Becerril, Elizabeth Monreal‐Escalante, Luis Hernández‐Adame, Miriam Angulo, José M. Mazon‐Suastegui
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Contribution of yeast models to virus research
R Sahaya Glingston, Jyoti Yadav, Jitika Rajpoot, Neha Joshi, Shirisha Nagotu
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Yarrowia lipolytica, health benefits for animals
Francisco A. Guardiola, María Ángeles Esteban, Carlos Angulo
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Isabel Bandín, Sandra Souto
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FEMS Yeast Research.2020;[Epub] CrossRef
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Ravinder Kumar, Piyush Kumar
FEMS Yeast Research.2019;[Epub] CrossRef
Development of conditional cell lysis mutants of Saccharomyces cerevisiae as production hosts by modulating OCH1 and CHS3 expression
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Vaccination with UV-inactivated nodavirus partly protects European sea bass against infection, while inducing few changes in immunity
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Research Support, Non-U.S. Gov't

Phosphorylation of the nucleocapsid protein of Hantaan virus by casein kinase II: Jeong-Joong Yoon , Yun-Tai Lee , Hin Chu , Seung-yeol Son , Manbok Kim; J. Microbiol. 2015;53(5):343-347. Published online May 3, 2015; DOI: https://doi.org/10.1007/s12275-015-5095-3

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Abstract

Hantaanvirus (HTNV) is the prototype of the genus Hantavirus, which belongs to the family Bunyaviridae. Hantaviruses are carried and transmitted by rodents and are known to cause two serious disease syndromes in humans i.e., hemorrhagic fever with renal syndrome (HFRS) and the hantavirus pulmonary syndrome (HPS). HTNV is an enveloped virus that contains a tripartite genome consisting of three negative-sense RNA segments (L, M, S), and the S and M segment of HTNV, respectively, encode the viral nucleocapsid protein (NP) and envelope glycoproteins. Possible phosphorylation motifs of casein kinase II (CKII) and protein kinase C (PKC) were identified in HTNV NP through bioinformatics searches. Sucrose gradient SDS-PAGE analysis indicated that dephosphorylated HTNV NP migrated faster than non-dephosphorylated NP, suggesting that HTNV NP is phosphorylated in infected Vero E6 cells. Immunoblot anaylsis of HTNV particles with anti-phosphoserine antibody and anti-phosphothreonine antibody after immunoprecipitation showed that viral particles are readily phosphorylated at threonine residues. In vitro kinase assay further showed that HTNV NP is phosphorylated by CK II, but not by PKC. Full length or truncated HTNV NPs expressed in E. coli were phosphorylated in vitro by CKII suggesting that phosphorylation may occur in vivo at multiple sites. Site specific mutagenesis studies suggest that HTNV NP phosphorylation might occur at unknown sites excluding the site-directly mutagenized locations. Taken together, HTNV NP can be phosphorylated mainly at threonine residues in vivo by CK II treatment.

Citations

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Protein kinase CK2: a potential therapeutic target for diverse human diseases
Christian Borgo, Claudio D’Amore, Stefania Sarno, Mauro Salvi, Maria Ruzzene
Signal Transduction and Targeted Therapy.2021;[Epub] CrossRef
Unique Interferon Pathway Regulation by the Andes Virus Nucleocapsid Protein Is Conferred by Phosphorylation of Serine 386
Matthew J. Simons, Elena E. Gorbunova, Erich R. Mackow, Susana López
Journal of Virology.2019;[Epub] CrossRef

Validation Study

Development and Validation of a Recombinant Nucleocapsid Protein-Based ELISA for Detection of the Antibody to Porcine Reproductive and Respiratory Syndrome Virus: Jia-Qi Chu , Xu-Min Hu , Myung-Cheol Kim , Chang-Sik Park , Moo-Hyung Jun; J. Microbiol. 2009;47(5):582-588. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0033-x

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Abstract: Three indirect enzyme-linked immunosorbent assays (iELISA) based on the North American like (NA-like), European like (EU-like) and co-expressed NA- and EU-like recombinant nucleocapsid proteins (N-protein) of porcine reproductive and respiratory syndrome virus (PRRSV) were validated for the detection of the antibodies in porcine sera. A total of 422 serum samples from unvaccinated pigs were tested. The cut-off value was optimized by a two-graph receiver operating characteristics analysis at a 95% confidence level. This assay was validated with Western blot analysis and IDEXX HerdChek™ ELISA. Cross-reactivity results showed that iELISA was PRRSV-specific. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs were less than 10%. The results indicate that iELISA is simpler to produce and perform, time-saving and suitable for large scale surveys of PRRSV infection at low cost, and is potentially useful to evaluate the efficiency of various vaccines against PRRSV.

Journal Articles

Nucleocapsid Amino Acids 211 to 254, in Particular, Tetrad Glutamines, are Essential for the Interaction Between the Nucleocapsid and Membrane Proteins of SARS-Associated Coronavirus: Xiaonan Fang , Lin-Bai Ye , Yijuan Zhang , Baozong Li , Shanshan Li , Lingbao Kong , Yuhua Wang , Hong Zheng , Wei Wang , Zhenghui Wu; J. Microbiol. 2006;44(5):577-580.; DOI: https://doi.org/2437 [pii]

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Abstract: GST pull-down assays were used to characterize the SARS-CoV membrane (M) and nucleocapsid (N) interaction, and it was found that the amino acids 211-254 of N protein were essential for this interaction. When tetrad glutamines (Q) were replaced with glutamic acids (E) at positions of 240-243 of the N protein, the interaction was disrupted.

Analysis of Immune Responses Against Nucleocapsid Protein of the Hantaan Virus Elicited by Virus Infection or DNA Vaccination: Gyu-Jin Woo , Eun-Young Chun , Keun Hee Kim , Wankee Kim; J. Microbiol. 2005;43(6):537-545.; DOI: https://doi.org/2292 [pii]

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Abstract: Even though neutralizing antibodies against the Hantaan virus (HTNV) has been proven to be critical against viral infections, the cellular immune responses to HTNV are also assumed to be important for viral clearance. In this report, we have examined the cellular and humoral immune responses against the HTNV nucleocapsid protein (NP) elicited by virus infection or DNA vaccination. To examine the cellular immune response against HTNV NP, we used H-2Kb restricted T-cell epitopes of NP. The NP-specific CD8+ T cell response was analyzed using a 51Cr-release assay, intracellular cytokine staining assay, enzyme-linked immunospot assay and tetramer binding assay in C57BL/6 mice infected with HTNV. Using these methods, we found that HTNV infection elicited a strong NP-specific CD8+ T cell response at eight days after infection. We also found that several different methods to check the NP-specific CD8+ T cell response showed a very high correlation among analysis. In the case of DNA vaccination by plasmid encoding nucleocapsid gene, the NP-specific antibody response was elicited 2 ~ 4 weeks after immunization and maximized at 6~8 weeks. NP-specific CD8+ T cell response reached its peak 3 weeks after immunization. In a challenge test with the recombinant vaccinia virus expressing NP (rVV-HTNV-N), the rVV-HTNV-N titers in DNA vaccinated mice were decreased about 100-fold compared to the negative control mice.

Production and Prophylactic Efficacy Study of Human Papillomavirus-like Particle Expressing HPV16 L1 Capsid Protein: Jie-Yun Park , Hyun-Mi Pyo , Sun-Woo Yoon , Sun-Young Baek , Sue-Nie Park , Chul-Joong Kim , Haryoung Poo; J. Microbiol. 2002;40(4):313-318.

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Abstract: To perform the prophylactic study of a vaccine derived from human papillomavirus (HPV) using Balb/c mice, we produced virus like particles consisting of HPV capsid protein L1 which has been reported to induce significant humoral and cellular immunity using various animal model systems. In order to produce HPV16 VLPs, the cDNA of L1 capsid protein in HPV type 16, obtained by polymerase chain reaction, was inserted into yeast expression vector, YEG[alpha]-HIR525 under the control of GAL10 promoter. The transformation of YEG[alpha]-HPV16 L1 was performed into the yeast Saccharomyces cerevisiae Y2805 by the lithium acetate method and the yeast clone expressing the highest level of L1 capsid protein of human papillomavirus type 16 was selected by Western blot analysis using anti-HPV16 L1 antibody. The purification of HPV16 VLP has been performed by the ultracentrifugation and gel-filtration methods. To validate the vaccine efficacy of the purified HPV16 VLPs and investigate the properties of HPV16 VLPs to induce humoral immunity, ELISA assay was performed. A significantly increased production of anti-HPV16 VLP antibodies was observed in sera from immunized mice. The neutralization activity of antibodies in the sera from the vaccinated mice was demonstrated by a rapid and simple assay to detect hemagglutihation inhibition activity

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