Table 1.
Sequences and purpose of the primers used in this work
| Primera | Sequence (5′ to 3′)b | Purpose |
|---|---|---|
| B2-For | GAATTCATGAACGACAACCAGAAAC (EcoRI) | Construction of B2-encoded plasmids for prokaryotic expression and eukaryotic transient expression |
| B2-Rev | TCTAGAGAGTTTCGATGGGTCTGCC (XbaI) | |
| B2ΔN20- For | GAATTCACCATGGCGACCCGAACGCTGGT (EcoRI) | |
| B2ΔN20-Rev 1 | TCTAGAGAGTTTCGATGGGTCTGCC (XbaI) | |
| B2ΔN20-Rev 2 | CTCGAGTTAGAGTTTCGATGGGTCTGCC (XhoI) | |
| B2R17A- For | GAATTCACCATGGCGAACGACAACCAGAAACAAGCATTGACAGCGTACCAAGCACTGGTCgccCGCCAAGT (EcoRI) | |
| B2R17A-Rev | TCTAGAGAGTTTCGATGGGTCTGCC (XbaI) | |
| B2R18A- For | GAATTCACCATGGCGAACGACAACCAGAAACAAGCATTGACAGCGTACCAAGCACTGGTCCGCgccCAAGTAAT (EcoRI) | |
| B2R18A-Rev | TCTAGAGAGTTTCGATGGGTCTGCC (XbaI) | |
| B2R24A- For | GAATTCACCATGGCGAACGACAACCAGAAACAAGCATTGACAGCGTACCAAGCACTGGTCCGCCGCCAAGTAATGGCGACCgccACG (EcoRI) | |
| B2R24A-Rev | TCTAGAGAGTTTCGATGGGTCTGCC (XbaI) | |
| B2R40A- For* | TCAAAAGGCAAGCGGAgccCTAAAGGCC | |
| B2R40A-Rev* | TAGggcCCCGCTTGCCTTTTGAAGGGCC | |
| B2R58A- For* | CGATCCCGCCACACTTCGTCTCAgccAT | |
| B2R58A-Rev* | CATggcTGAGACGAAGTGTGGCGGGATC | |
| B2K6A- For | GAATTCACCATGGCGAACGACAACCAGgccCAAGC (EcoRI) | |
| B2K6A-Rev | TCTAGAGAGTTTCGATGGGTCTGCC (XbaI) | |
| B2K36A- For* | CAAgccGCAAGCGGAAGGCTAAAGGC | |
| B2K36A-Rev* | CTTTAGCCTTCCGCTTGCggcTT | |
| B2K42A- For* | TAgccGCCGTAGGCCTCGAACCGC | |
| B2K42A-Rev* | GCGGTTCGAGGCCTACGGCggcTAGCCTT | |
| B2K75A- For | GAATTCACCATGGCGAACGACAACCAGAAAC (EcoRI) | |
| B2K75A-Rev | TCTAGAGAGTTTCGATGGGTCTGCCTCCTCGCTTGGggcCG (XbaI) | |
| B2K84A- For | GAATTCACCATGGCGAACGACAACCAGAAAC (EcoRI) | |
| B2K84A-Rev | TCTAGAGAGggcCGATGGGTCTGCC (XbaI) | |
| siRNA-EGFP | Chemical synthesis of siRNAs that induce RNA interference in Pr-E cells | |
| Homologous sequence | AAGCTGACCCTGAAGTTCATC | |
| Sense strand | p-GCUGACCCUGAAGUUCAUCUU | |
| Antisense strand | UUCGACUGGGACUUCAAGUAG-p | |
| siRNA-Fluc | ||
| Homologous sequence | GATTATGTCCGGTTATGTA | |
| Sense strand | p-GAUUAUGUCCGGUUAUGUAUU | |
| Antisense strand | UUCUAAUACAGGCCAAUACAU-p | |
| Anti EGFP-For | GATCCGCCACAACATCGAGGACGGC | Construction of in vitro- transcription templates for preparing probes and dsRNA |
| Anti EGFP-Rev | TAATACGACTCACTATAGGTTACTTGTACAGCTCGTCCATGCC | |
| Anti siRNA-For | GAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATC | |
| Anti siRNA-Rev | TAATACGACTCACTATAGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTT | |
| Sense siRNA-For | TAATACGACTCACTATAGGGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATC | |
| Sense siRNA-Rev | CAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTT | |
| T7-dsRNA-For | TAATACGACTCACTATAGGATGGTGAGCTAGGGCGAGGA | |
| T7-dsRNA-Rev | TAATACGACTCACTATAGGTTGAAGTTCACCTTGATGCC |
The GenBank accession numbers of the B2 proteins used in the current study are listed in parentheses as follows: FHV (X77156), NoV (AAF97861), BBV (AAA42746), BoV (AAK15752), GGNNV (AAK21878), SJNNV (BAA84211), and RGNNV (AAX77408). Sequence-specific primers are designed according to GenBank accession numbers AY962576 (WhNV RNA1) and U55762 (EGFP), and primers with asterisks are designed for overlapping PCR.
Underlined characters indicate restriction endonuclease sites, and the types are shown in parentheses. Substituted nucleotides for mutagenesis are shown with lowercase characters. The T7 polymerase promoter is shown in italics. The EGFP ORF region is amplified from pEGFP-N1 (Clontech). The Kozak translation initiation sequence designed according to the pAc 5.1 (Invitrogen) handbook is indicated with boldfaced characters.