Table 1
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 |
[i] bp – base pairs
Table 2
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 |
[i] F – forward; R – reverse; IL – interleukin; INF-γ – interferon gamma
Table 3
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 |
[i] F – forward; R – reverse; IL – interleukin; INF-γ – interferon gamma
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×)
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
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
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
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
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)
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