Background
Methods
Cells and virus
Antibodies and reagents
Virus infection
Construction and screening of Suppression subtractive hybridization (SSH) library
qRT-PCR analysis of CypB expression in ORFV-infected MDBK cells
Western blotting analysis of CypB expression in ORFV-infected MDBK cells
Anti-ORFV activity of CsA in vitro detected by MTT assay
CypB gene silencing by siRNA
Name | Primer Sequences (5′-3′) | |
---|---|---|
CypB specific siRNA | up | GCAUCUACGGUGAACGCUUTT |
down | AAGCGUUCACCGUAGAUGCTT | |
Negative control siRNA | up | UUCUCCGAACGUGUCACGUTT |
down | ACGUGACACGUUCGGAGAATT |
Immunofluorescence assay after siRNA knockdown of CypB
Effect of CypB gene silencing on ORFV replication
Statistical analysis
Results
CypB was found to be significantly up-regulated in the ORFV-infected MDBK cells by SSH technique
EST No. | Species | Gene homology | Gene name | Fold upregulation |
---|---|---|---|---|
F-1 |
Bos Taurus
| Bos taurus beta-actin (ACTB) mRNA | ACTB | 1.5 |
F-2 |
Bos Taurus
| Bos taurus actin, gamma 1 (ACTG1), mRNA | ACTG1 | 1.7 |
F-3 | unknown | unknown | unknown | 3.4 |
F-4 |
Bos Taurus
| Bos taurus vacuole membrane protein 1 (VMP1), mRNA | VMP1 | 3.7 |
F-5 |
Bos Taurus
| Bos taurus ring finger protein 19B (RNF19B), mRNA | RNF19B | 1.9 |
F-6 |
Bos Taurus
| Bos taurus zinc finger protein 408 (ZNF408), mRNA | ZNF408 | 2.3 |
F-7 |
Bos Taurus
| Bos taurus zinc finger CCCH-type containing 14 (ZC3H14), transcript variant 1, mRNA | ZC3H14 | 1.5 |
F-8 |
Bos Taurus
| Bos taurus ATPase, Na+/K+ transporting, beta 1 polypeptide (ATP1B1), mRNA. | ATP1B1 | 3.4 |
F-9 |
Bos Taurus
| Bos taurus poly-U binding splicing factor 60KDa (PUF60), mRNA. | PUF60 | 2.3 |
F-10 | unknown | unknown | unknown | 2.6 |
F-11 |
Bos Taurus
| Bos taurus eukaryotic translation initiation factor 3, subunit L (EIF3L), mRNA | EIF3L | 3.7 |
F-12 |
Bos Taurus
| Bos taurus eukaryotic translation elongation factor 1 alpha 1 EEF1A1), mRNA | EEF1A1 | 3.4 |
F-13 |
Bos Taurus
| Bos taurus eukaryotic translation initiation factor 5 (EIF5), mRNA | EIF5 | 2.9 |
F-14 |
Bos Taurus
| Bos taurus cytohesin 2 (CYTH2), mRNA | CYTH2 | 1.6 |
F-15 |
Bos Taurus
| Bos taurus transmembrane emp24 protein transport domain containing 1 (TMED1), mRNA | TMED1 | 3.6 |
F-16 |
Bos Taurus
| Bos taurus transmembrane protein 179B (TMEM179B), mRNA | TMEM179B | 2.8 |
F-17 |
Bos Taurus
| Bos taurus neuropilin 2 (NRP2), mRNA | NRP2 | 2.1 |
F-18 |
Bos Taurus
| Sin3A-associated protein, 18 kDa (SAP18), mRNA | SAP18 | 2.4 |
F-19 |
Bos Taurus
| Bos taurus renin binding protein (RENBP), mRNA | RENBP | 1.3 |
F-20 |
Bos Taurus
| Bos taurus signal sequence receptor, alpha (SSR1), mRNA | SSR1 | 1.9 |
F-21 |
Bos Taurus
| Bos taurus mitogen-activated protein kinase kinase 6 (MAP2K6), mRNA | MAP2K6/MKK6 | 9.2 |
F-22 |
Bos Taurus
| Bos Taurus solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, system Xag), member 1 (SLC1A1), mRNA. | SLC1A1 | 2.7 |
F-23 |
Bos Taurus
| Bos taurus isolate Mcg489 mitochondrion, complete genome | Mcg489 mitochondrion | 2.5 |
F-24 |
Bos Taurus
| Calmodulin 1 (phosphorylase kinase, delta) (CALM1), mRNA | CALM1 | 2.3 |
F-25 |
Bos Taurus
| Bos taurus enhancer of rudimentary homolog (Drosophila) (ERH), mRNA | ERH | 1.7 |
F-26 |
Bos Taurus
| Bos taurus threonyl-tRNA synthetase-like 1 (TARSL1), mRNA, complete cds. | TARSL1 | 2.1 |
F-27 |
Bos Taurus
| Bos taurus threonyl-tRNA synthetase 2, mitochondrial (putative) (TARS2), mRNA | TARS2 | 1.8 |
F-28 |
Bos Taurus
| Bos taurus S100 calcium binding protein A2 (S100A2), mRNA | S100A2 | 9.7 |
F-29 |
Bos Taurus
| Bos taurus lactate dehydrogenase B (LDHB), mRNA, complete cds. | LDHB | 3.5 |
F-30 |
Bos Taurus
| Bos taurus H3 histone, family 3A (H3F3A), mRNA. | H3F3A | 4.2 |
F-31 |
Bos Taurus
| Bos taurus heterogeneous nuclear ribonucleoprotein U (scaffold attachment factor A) (HNRNPU), mRNA. | HNRNPU | 2.9 |
F-32 |
Bos Taurus
| Bos taurus RAB2A, member RAS oncogene family (RAB2A), mRNA. | RAB2A | 1.5 |
F-33 |
Bos Taurus
| Bos taurus alanyl-tRNA synthetase (AARS), mRNA | AARS | 1.9 |
F-34 |
Bos Taurus
| Bos taurus minichromosome maintenance complex component 4 (MCM4), mRNA | MCM4 | 2.7 |
F-35 |
Bos Taurus
| Bos taurus tubulin, beta 6, mRNA | TUBB6 | 1.3 |
F-36 |
Bos Taurus
| Bos taurus tubulin, alpha 1c (TUBA1C), mRNA | TUBA1C | 1.7 |
F-37 |
Bos Taurus
| Bos taurus tubulin, alpha 1b (TUBA1B), mRNA | TUBA1B | 1.2 |
F-38 |
Bos Taurus
| Bos taurus tropomyosin 1 (alpha) (TPM1), mRNA | TPM1 | 2.0 |
F-39 |
Bos Taurus
| Bos taurus tropomyosin 4 (TPM4), mRNA | TPM4 | 1.5 |
F-40 |
Bos Taurus
| Bos taurus minichromosome maintenance complex component 6 (MCM6), mRNA | MCM6 | 2.3 |
F-41 |
Bos Taurus
| Bos taurus chromosome 10 open reading frame, human C14orf1 (C10H14orf1), mRNA. | C10H14orf1 | 2.4 |
F-42 |
Bos Taurus
| Bos taurus CLPTM1-like (CLPTM1L), mRNA | CLPTM1L | 4.3 |
F-43 |
Bos Taurus
| Bos taurus CD151 molecule (Raph blood group) (CD151), mRNA | CD151 | 7.6 |
F-44 |
Bos Taurus
| Bos taurus ubiquinol-cytochrome c reductase binding protein (UQCRB), mRNA. | UQCRB | 2.6 |
F-45 |
Bos Taurus
| Bos taurus low molecular mass ubiquinone-binding protein mRNA, complete cds. | Low molecular mass ubiquinone-binding protein | 2.1 |
F-46 |
Bos Taurus
| Bos taurus dynein, cytoplasmic 1, light intermediate chain 1 (DYNC1LI1), mRNA | DYNC1LI1 | 3.5 |
F-47 |
Bos Taurus
| Bos taurus glucose-6-phosphate isomerase (GPI), mRNA | GPI | 2.9 |
F-48 |
Bos Taurus
| Bos taurus galactosidase, beta 1 (GLB1), mRNA | GLB1 | 2.4 |
F-49 |
Bos Taurus
| Bos taurus proteasome (prosome, macropain) activator subunit 1 (PA28 alpha) (PSME1), mRNA. | PSME1 | 1.3 |
F-50 |
Bos Taurus
| Bos taurus peptidylprolyl isomerase B (cyclophilin B) (PPIB), mRNA | PPIB | 10.2 |
F-51 |
Bos Taurus
| Bos taurus peroxiredoxin 2 (PRDX2), mRNA. | PRDX2 | 3.4 |
F-52 |
Bos Taurus
| Bos taurus proteolipid protein 2 (colonic epithelium-enriched) (PLP2), mRNA. | PLP2 | 3.7 |
F-53 |
Bos Taurus
| Bos taurus voucher proven bull 27,223 mitochondrion, complete genome. | Bos taurus voucher proven bull 27,223 mitochondrion | 2.1 |
F-54 |
Bos Taurus
| Bos taurus Fas-activated serine/threonine kinase (FASTK), mRNA. | FASTK | 4.6 |
F-55 |
Bos Taurus
| Bos taurus ferritin, light polypeptide (FTL), mRNA | FTL | 2.5 |
F-56 |
Bos Taurus
| Bos taurus Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) ubiquitously expressed (FAU), mRNA. | FAU | 1.2 |
F-57 |
Bos Taurus
| Bos taurus acyl-CoA thioesterase 7 (ACOT7), mRNA | ACOT7 | 1.9 |
F-58 |
Bos Taurus
| Bos taurus adenine phosphoribosyltransferase (APRT), mRNA | APRT | 1.5 |
F-59 |
Bos Taurus
| Bos taurus mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (MGAT1), mRNA, complete cds | MGAT1 | 1.7 |
F-60 |
Bos Taurus
| Bos taurus lactate dehydrogenase C (LDHC), transcript variant 1, mRNA. | LDHC | 2.3 |
EST No. | Species | Gene homology | Gene name | Fold downregulation |
---|---|---|---|---|
R-1 |
Bos Taurus
| Bos taurus mitogen-activated protein kinase kinase 4 (MAP2K4), mRNA. | MAP2K4/MKK4 | 8.7 |
R-2 |
Bos Taurus
| Bos taurus neuroplastin (NPTN), mRNA | NPTN | 1.9 |
R-3 |
Bos Taurus
| Bos taurus nuclear transport factor 2 (NUTF2), mRNA | NUTF2 | 2.5 |
R-4 |
Bos Taurus
| Bos taurus S100 calcium binding protein A4 (S100A4), mRNA | S100A4 | 9.6 |
R-5 |
Bos Taurus
| Bos taurus acyl-CoA synthetase long-chain family member 5 (ACSL5), mRNA, complete cds | ACSL5 | 2.9 |
R-6 |
Bos Taurus
| Bos taurus eukaryotic translation elongation factor 2 (EEF2), mRNA | EEF2 | 3.8 |
R-7 |
Bos Taurus
| Bos taurus poly-U binding splicing factor 60KDa (PUF60), mRNA | PUF60 | 1.3 |
R-8 |
Bos Taurus
| Bos taurus H3 histone, family 3B (H3.3B) (H3F3B), mRNA | H3F3B | 2.6 |
R-9 |
Bos Taurus
| Bos taurus glyceraldehyde-3-phosphate dehydrogenase (GAPDH), mRNA | GAPDH | 1.2 |
R-10 |
Bos Taurus
| Bos taurus NADH dehydrogenase (ubiquinone) complex I, assembly factor 1 (NDUFAF1), mRNA | NDUFAF1 | 1.7 |
R-11 |
Bos Taurus
| Bos taurus solute carrier family 51, beta subunit (SLC51B), mRNA | SLC51B | 3.2 |
R-12 |
Bos Taurus
| Bos taurus ferredoxin 1 (FDX1), mRNA | FDX1 | 2.5 |
R-13 |
Bos Taurus
| Bos taurus transforming growth factor beta 1 induced transcript 1 (TGFB1I1), mRNA | TGFB1I1 | 1.4 |
R-14 |
Bos Taurus
| Bos taurus heat shock 70 kDa protein 8 (HSPA8), mRNA | HSPA8 | 10.2 |
R-15 |
Bos Taurus
| Bos taurus vimentin (VIM), mRNA | VIM | 6.4 |
R-16 |
Bos Taurus
| Bos taurus lectin, galactoside-binding, soluble, 1 (LGALS1), mRNA | LGALS1 | 2.1 |
R-17 |
Bos Taurus
| Bos taurus clone IMAGE:7,961,517 NDP kinase NBR-A mRNA, complete cds | NDP kinase NBR-A | 1.5 |
R-18 |
Bos Taurus
| Bos taurus polymerase (RNA) I polypeptide D (POLR1D), mRNA | POLR1D | 1.5 |
R-19 |
Bos Taurus
| Bos taurus guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 1 (GNAI1), mRNA | GNAI1 | 1.2 |
R-20 |
Bos Taurus
| Bos taurus annexin A2 (ANXA2), mRNA | ANXA2 | 1.9 |
R-21 |
Bos Taurus
| Bos taurus Williams Beuren syndrome chromosome region 22 (WBSCR22), mRNA | WBSCR22 | 2.3 |