Production and Characterization of Self-Assembled Virus-like Particles Comprising Capsid Proteins from Genotypes 3 and 4 Hepatitis E Virus (HEV) and Rabbit HEV Expressed in Escherichia coli
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plasmid Construction and Truncated HEV ORF2 Protein Expression
2.2. Purification of Recombinant HEV ORF2 Proteins
2.3. SDS-PAGE and Western Blot Analysis
2.4. Analysis of Purified HEV VLPs with the Bioanalyzer
2.5. Transmission Electron Microscopy (TEM)
2.6. Immunoelectron Microscopy (IEM)
2.7. Enzyme-Linked Immunosorbent Assays (ELISAs) for the Determination of Binding with Monoclonal Antibodies (MAbs) against ORF2 Protein of Human HEV (HEV-3 and HEV-4), Rabbit HEV (HEV-3ra), or Rat HEV (HEV-C1)
2.8. Immunization of HEV VLPs in Mice
3. Results
3.1. The Expression and Purification of Truncated HEV ORF2 Proteins
3.2. Characterization of the Purified VLPs of HEV-3, HEV-3ra, and HEV-4
3.3. The Antigenicity of the Purified VLPs of HEV-3, HEV-3ra, and HEV-4
3.4. Immune Response of the Balb/cAJcl Mice to the Purified VLPs of HEV-3, HEV-3ra, and HEV-4
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Primer Name | Sequence (5′–3′) | Note |
---|---|---|
hHEV_G3 Nde-ORF2 aa12 Fw | CATATG GTGCTTCTGCCTATGCTGCC | Nde I site (underlined) |
hHEV_G3 Nde-ORF2 aa14 Fw | CATATG CTGCCTATGCTGCCCGCGCC | Nde I site (underlined) |
hHEV_G3 Nde-ORF2 aa61 Fw | CATATG CCCTTTGCCGCCGATGTCG | Nde I site (underlined) |
hHEV_G3 Nde-ORF2 aa89 Fw | CATATG GACCAGTCCCAGCGCCCCTC | Nde I site (underlined) |
hHEV_G3 Nde-ORF2 aa107 Fw | CATATG GCTGCGCCGTTGACTGCTATC | Nde I site (underlined) |
hHEV_G3 Nde-ORF2 aa112 Fw | CATATG GCTATCTCACCAGCCCCTGAC | Nde I site (underlined) |
hHEV_G3 Nde-ORF2 aa117 Fw | CATATG CCTGACACAGCCCCTGTACC | Nde I site (underlined) |
hHEV_G3 Nde-ORF2 aa122 Fw | CATATG GTACCTGATGTTGATTCACG | Nde I site (underlined) |
hHEV_G3 Nde-ORF2 aa368 Fw | CATATG ATTGCTCTGACACTGTTCAAC | Nde I site (underlined) |
rbHEV_G3 Nde-ORF2 aa112 Fw | CATATG GCTGTTTCACCTGCACCTG | Nde I site (underlined) |
rbHEV_G3 Nde-ORF2 aa368 Fw | CATATG ATAGCCCTGACGCTGTTTAAC | Nde I site (underlined) |
hHEV_G4 Nde-ORF2 aa112 Fw | CATATG GCTGTGGCCCCGGCCCCCGA | Nde I site (underlined) |
hHEV_G4 Nde-ORF2 aa114 Fw | CATATG GCCCCGGCCCCCGATACTGC | Nde I site (underlined) |
hHEV_G4 Nde-ORF2 aa117 Fw | CATATG CCCGATACTGCTCCTGTTCC | Nde I site (underlined) |
hHEV_G4 Nde-ORF2 aa366 Fw | CATATG GTCGGTCGTGGTATAGCGC | Nde I site (underlined) |
hHEV_G4 Nde-ORF2 aa368 Fw | CATATG CGTGGTATAGCGCTAACTCTG | Nde I site (underlined) |
hHEV_G4 Nde-ORF2 aa370 Fw | CATATG ATAGCGCTAACTCTGTTCAATC | Nde I site (underlined) |
hHEV_G3 ORF2-Bam aa606 Rev | GGATCC CTA AGCCGAGTGCGGGGCTAGTAC | BamH I site (underlined) |
rbHEV_G3 ORF2-Bam aa606 Rev | GGATCC CTA AACTGAGTGCGGAGCAAGC | BamH I site (underlined) |
hHEV_G4 ORF2-Bam aa606 Rev | GGATCC CTA CGCAGAGTGAGGTGCGAGGAC | BamH I site (underlined) |
hHEV_G4 ORF2-Bam aa609 Rev | GGATCC CTA AGCGGCCAGCGCAGAGTGAG | BamH I site (underlined) |
hHEV_G4 ORF2-Bam aa612 Rev | GGATCC CTA GTCCTCTAAAGCGGCCAGCG | BamH I site (underlined) |
rbHEV_G3 T6686C Fw | TACGGCTCGTCAACTAACCC | nt 6684-6703 a (mutated nt underlined) |
rbHEV_G3 T6686C Rev | TGTAGTTTGATCATACTCTG | nt 6664-6683 a |
Purified VLPs | Amount (mL) Recovered after Gel Filtration | Concentration (Mean ± SD, mg/mL) a | Molecular Weight (Mean ± SD, kDa) b | Yield (Mean ± SD, mg) | Purity (Mean ± SD, %) b |
---|---|---|---|---|---|
HEV-3-VLP_89-606 (p518) | 48 | 0.5 ± 0.1 | 63.4 ± 0.7 | 24.0 ± 6.8 | 89.5 ± 2.8 |
HEV-3-VLP_107-606 (p500) | 48 | 1.4 ± 0.1 | 61.1 ± 0.4 | 66.0 ± 6.0 | 90.7 ± 4.1 |
HEV-3-VLP_112-606 (p495) | 48 | 1.9 ± 0.3 | 61.0 ± 0.5 | 89.6 ± 15.4 | 95.3 ± 1.4 |
HEV-3-VLP_117-606 (p490) | 48 | 1.3 ± 0.1 | 60.3 ± 0.6 | 64.0 ± 5.5 | 91.9 ± 2.3 |
HEV-3-VLP_122-606 (p485) | 48 | 1.6 ± 0.4 | 58.4 ± 1.1 | 76.8 ± 17.3 | 95.0 ± 1.6 |
HEV-3-VLP_368-606 (p239) | 12 | 1.4 ± 0.6 | 27.1 ± 0.5 | 16.2 ± 7.6 | 93.1 ± 0.1 |
HEV-3ra-VLP_112-606 (p495) | 16 | 0.9 ± 0.2 | 62.7 ± 0.6 | 13.9 ± 3.7 | 94.9 ± 1.9 |
HEV-4-VLP_114-606 (p493) | 48 | 0.8 ± 0.1 | 61.9 ± 0.7 | 39.2 ± 5.0 | 74.7 ± 5.8 |
HEV-4-VLP_117-606 (p490) | 48 | 1.3 ± 0.4 | 60.7 ± 0.3 | 62.4 ± 20.4 | 90.1 ± 8.5 |
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Kobayashi, T.; Takahashi, M.; Ohta, S.; Hoshino, Y.; Yamada, K.; Jirintai, S.; Primadharsini, P.P.; Nagashima, S.; Murata, K.; Okamoto, H. Production and Characterization of Self-Assembled Virus-like Particles Comprising Capsid Proteins from Genotypes 3 and 4 Hepatitis E Virus (HEV) and Rabbit HEV Expressed in Escherichia coli. Viruses 2024, 16, 1400. https://doi.org/10.3390/v16091400
Kobayashi T, Takahashi M, Ohta S, Hoshino Y, Yamada K, Jirintai S, Primadharsini PP, Nagashima S, Murata K, Okamoto H. Production and Characterization of Self-Assembled Virus-like Particles Comprising Capsid Proteins from Genotypes 3 and 4 Hepatitis E Virus (HEV) and Rabbit HEV Expressed in Escherichia coli. Viruses. 2024; 16(9):1400. https://doi.org/10.3390/v16091400
Chicago/Turabian StyleKobayashi, Tominari, Masaharu Takahashi, Satoshi Ohta, Yu Hoshino, Kentaro Yamada, Suljid Jirintai, Putu Prathiwi Primadharsini, Shigeo Nagashima, Kazumoto Murata, and Hiroaki Okamoto. 2024. "Production and Characterization of Self-Assembled Virus-like Particles Comprising Capsid Proteins from Genotypes 3 and 4 Hepatitis E Virus (HEV) and Rabbit HEV Expressed in Escherichia coli" Viruses 16, no. 9: 1400. https://doi.org/10.3390/v16091400