Open Access

Recombinant hexon protein as a new bovine adenovirus type 3 subunit vaccine candidate

Uudamsaikhan Gundegmaa
Uudamsaikhan Gundegmaa
, 
Odbileg Raadan
Odbileg Raadan
, 
Hsing-Chieh Wu
Hsing-Chieh Wu
, 
Hsian-Yu Wang
Hsian-Yu Wang
, 
Min-Chia Wu
Min-Chia Wu
 and 
Chun-Yen Chu
Chun-Yen Chu
   | Mar 17, 2023
Journal of Veterinary Research's Cover Image
About this article
Previous Article
Next Article
Cite
Published Online: Mar 17, 2023
Page range: 23 - 31
Received: Sep 05, 2022
Accepted: Mar 02, 2023
DOI: https://doi.org/10.2478/jvetres-2023-0014
Keywords
© 2023 U. Gundegmaa et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1

Bovine adenovirus 3 (BAdV-3) identification and confirmation (a) Gradient PCR for detection of BAdV-3. Lane N – negative control; lane 1 – annealing temperature 50°C; lane 2 – 50.5°C; lane 3 – 51°C; lane 4 – 52°C; lane M – marker (100 base pairs (bp) – 3,000 bp) (b) Transmission electron microscopy image of the typical virion approximately 75 nm in diameter. Phosphotungstic acid negative staining (100,000×)
Bovine adenovirus 3 (BAdV-3) identification and confirmation (a) Gradient PCR for detection of BAdV-3. Lane N – negative control; lane 1 – annealing temperature 50°C; lane 2 – 50.5°C; lane 3 – 51°C; lane 4 – 52°C; lane M – marker (100 base pairs (bp) – 3,000 bp) (b) Transmission electron microscopy image of the typical virion approximately 75 nm in diameter. Phosphotungstic acid negative staining (100,000×)

Fig. 2

Cloning and expression analysis of recombinant hexon protein of bovine adenovirus 3 (a) Expressed rhexon with 44 kDa molecular weight separated on 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis. Lane 1 –induction time 0 h; lane 2 – 2 h; lane 3 – 4 h; lane 4 – 6 h; lane M – marker (b) Western blot analysis with mouse anti-His antibody. Lane 1 – induction time 0 h; lane 2 – 2 h; lane 3 – 4 h; lane 4 – 6 h; lane M – marker
Cloning and expression analysis of recombinant hexon protein of bovine adenovirus 3 (a) Expressed rhexon with 44 kDa molecular weight separated on 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis. Lane 1 –induction time 0 h; lane 2 – 2 h; lane 3 – 4 h; lane 4 – 6 h; lane M – marker (b) Western blot analysis with mouse anti-His antibody. Lane 1 – induction time 0 h; lane 2 – 2 h; lane 3 – 4 h; lane 4 – 6 h; lane M – marker

Fig. 3

Antibody response in indirect ELISA in mice immunised with purified or unpurified rhexon protein. Total anti-bovine adenovirus 3 (BAdV-3) immunoglobulin G levels after inoculation with (a) 10 μg/mL of BAdV-3 and (b) 1.25 μg/mL of rhexon protein. Ag – antigen; OD – optical density; a, b – significant difference (P < 0.05). Data represent means ± standard deviation
Antibody response in indirect ELISA in mice immunised with purified or unpurified rhexon protein. Total anti-bovine adenovirus 3 (BAdV-3) immunoglobulin G levels after inoculation with (a) 10 μg/mL of BAdV-3 and (b) 1.25 μg/mL of rhexon protein. Ag – antigen; OD – optical density; a, b – significant difference (P < 0.05). Data represent means ± standard deviation

Fig. 4

Antibody response in indirect ELISA in mice immunised with different amounts of rhexon protein. Total anti-bovine adenovirus 3 (BAdV-3) immunoglobulin G levels after inoculation with (a) 10 μg/mL of BAdv-3 and (b) 1.25 μg/mL of rhexon protein. Ag – antigen; OD – optical density; a, b, c – significant difference (P < 0.05). Data represent means ± standard deviation
Antibody response in indirect ELISA in mice immunised with different amounts of rhexon protein. Total anti-bovine adenovirus 3 (BAdV-3) immunoglobulin G levels after inoculation with (a) 10 μg/mL of BAdv-3 and (b) 1.25 μg/mL of rhexon protein. Ag – antigen; OD – optical density; a, b, c – significant difference (P < 0.05). Data represent means ± standard deviation

Fig. 5

Long-term antibody response in indirect ELISA in mice immunised with rhexon protein. Total anti-bovine adenovirus 3 (BAdV-3) immunoglobulin G levels after inoculation with (a) 10 μg/mL of BAdV-3 and (b) 1.25 μg/mL of rhexon protein. Ag – antigen; OD – optical density; a, b – significant difference (P < 0.05). Data represent means ± standard deviation
Long-term antibody response in indirect ELISA in mice immunised with rhexon protein. Total anti-bovine adenovirus 3 (BAdV-3) immunoglobulin G levels after inoculation with (a) 10 μg/mL of BAdV-3 and (b) 1.25 μg/mL of rhexon protein. Ag – antigen; OD – optical density; a, b – significant difference (P < 0.05). Data represent means ± standard deviation

Fig. 6

Cytokine response in immunised mice. The mRNA expression levels of interleukin (IL)-2, IL-6, IL-12 and interferon gamma (INF-γ) are shown as fold change relative to the saline control at two and four weeks post immunisation. a, b, c – significant difference (P < 0.05)
Cytokine response in immunised mice. The mRNA expression levels of interleukin (IL)-2, IL-6, IL-12 and interferon gamma (INF-γ) are shown as fold change relative to the saline control at two and four weeks post immunisation. a, b, c – significant difference (P < 0.05)

Fig. 7

Cytokine levels and antibody response in goats immunised with rhexon protein(a) – goat mRNA expression levels of interleukin (IL)-21, IL-6, IL-12 and interferon gamma (INF-γ) are shown as fold change relative to the saline control at two and four weeks post immunisation (b) indirect ELISA with 1.25 μg/mL of rhexon protein. a, b – significant difference (P < 0.05) Ag – antigen; OD – optical density. Data represent means ± standard deviation
Cytokine levels and antibody response in goats immunised with rhexon protein(a) – goat mRNA expression levels of interleukin (IL)-21, IL-6, IL-12 and interferon gamma (INF-γ) are shown as fold change relative to the saline control at two and four weeks post immunisation (b) indirect ELISA with 1.25 μg/mL of rhexon protein. a, b – significant difference (P < 0.05) Ag – antigen; OD – optical density. Data represent means ± standard deviation

The sequence of primers used for PCR identification

Primer name Position Sequence (5′-3′) Size (bp) Reference
BALF 19235–19256 GRTGGTCIYTRGATRTRATGGA 641 (18)
BARF 19852–19872 AAGYCTRTCATCYCCDGGCCA
cBAdVH-F 1645–1659 AATGAGCTCCTTCAGAGCACTCTG 693 K01264.1
cBAdVH-R 2319–2337 ACTGCGGCCGCAGTTTCTATGGTTCAC GenBank

Sequences of primers for mouse cytokines and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in real-time PCR

Gene Sequence (5′ ̶ 3′) GenBank accession no.
GAPDH F : TCA ACA GCA ACT CCC ACT CTT CCA GU214026.1
R : ACC CTG TTG CTG TAG CCG TAT TCA
IL-2 F : CCT GAG CAG GGA GAA TTA CA AH001969.2
R : TCC AGA ACA TGC CGC AGA
IL-6 F : GAG GAT ACC ACT CCC AAC AG CC M20572.1
R : AAG TGC ATC ATC GTT GTT CAT ACA
IL-12 F : GAG CAC TCC CCA TTC CTA CT M86671.1
R : GCA TTC GAC TTC GGT AGA TG
INF-γ F : GGC CAT CAG CAA CAT AAG GGT AY423847.1
R : TCG GTT GTT GAC CTC AAA CTT GGC

Sequences of primers for goat cytokines and β-actin in real-time PCR

Gene Sequence (5′ ̶ 3′) GenBank accession no.
β-actin F : CCT TTT ACA ACG AGC TGC GTG TG AH00130
R : ACG TAG CAG AGC TTC TCC TTG ATG
INF-γ F : TTC AGA GCC AAA TTG TCT CC M29867
R : CTG GAT CTG CAG ATC ATC CA
IL-6 F : TCA TTA AGC GCA TGG TGG ACA AA NM173923
R : TCA GCT TAT TTT CTG CCA GTG TCT
IL-12 F : TTA TTG AGG TCG TGG TAG AAG CTG U11815
R : GGT CTC AGT TGC AGG TTC TTG G
IL-21 F : CAG TGG CCC ATA AGT CAA GC AB073021
R : TAC ATC TTC TGG AGC TGG CA
eISSN:
2450-8608
Language:
English
Publication timeframe:
4 times per year
Journal Subjects:
Life Sciences, Molecular Biology, Microbiology and Virology, other, Medicine, Veterinary Medicine
Journal RSS Feed