Syncrip facilitates porcine parvovirus viral dna replication through the alternative splicing of ns1 mrna to promote ns2 mrna formation

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Chuyển đổi dữ liệu	07.01.2023
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PPV.NS

Table 1 Primers used in this study Restriction enzyme sequences are underlined. Primers Sequences (5 ′–3 ′) Vectors
CTC GAG
GCG GCC GC
GTC GAC

RNA‑pulldown assay
RNA-pulldown assay was carried out as previously
described [
33
]. Briefly, NS1 sequences were T7 transcrip-
tion synthesized with biotinylation at 5′ end. 20 μL of
streptavidin C1 was used for preclear nuclear extract in
each sample for 30 min at 4 ℃. Then they were centri-
fuged at 1000 g for 5 min. The supernatant was collected
and supplemented with yeast tRNA (0.1 μg/μL), 20 μg
biotinylated NS1 mRNA or LacZ mRNA for 60 min at
4 ℃. 30 μL of streptavidin C1 was added to isolate RNA
Table 1 Primers used in this study
Restriction enzyme sequences are underlined.
Primers
Sequences (5′–3′)
Vectors
NS1-F
CACTC GAG ATG GAT TAC AAG GAT GAC GAC GAT AAG GCA GCG GGA AAC ACT TACTC
pCI-neo
NS1-R
GGC TCT AGA TTA TTC AAG GTT TGT TGT GGGTG
p-NS1-F
CACTC GAG ATG GCA GCG GGA AAC ACT TAC
pEGFP-N1
p-NS1-R
GGC GAA TTC GCA TTC AAG GTT TGT TGT GGGTG
NS2-F
CACTC GAG ATG GCA GCG GGA AAC ACT TAC
pEGFP-N1
NS2-R
GTC GGA TCC GTC CAA AGC AGG CTC TTA TG
p-NS2-F
CAGCT AGC ATG GCA GCG GGA AAC ACT TAC
pCDH-EF1-
MCS-T2A-
Puro
p-NS2-R
GTC GCG GCC GCGTC CAA AGC AGG CTC TTA TG
VP1-F
CACTC GAG ATG GCG CCT CCT GCA AAA AGA GCA AGA GGA CTA ACT CTA CCA GGA TAC
pEGFP-N1
VP1-R
GTC GGA TCC GTG TAT AAT TTT CTT GGT ATAAG
VP2-F
CACTC GAG ATG AGT GAA AAT GTG GAA C
pEGFP-N1
VP2-R
GTC GGA TCC GTG TAT AAT TTT CTT GGT ATAAG
SYNCRIP-F
GGC GTC GAC TCA TGG CTA CAG AAC ATG TTA ATG GA
pGEX-4 T-1
SYNCRIP-R
GGC GCG GCC GCTTA TTG TAA CAG GTC AGG ACCGG
p-SYNCRIP-F
GGC CTC GAG ATG GCT ACA GAA CAT GTT AAT GGA
pCDNA3.0
p-SYNCRIP-R
GGC TCT AGA TTA ATG GTG ATG GTG ATG ATG GGT GGA GGC TTG TAA CAG GTC AGG ACC GG

Page 5 of 15
Chen et al. Vet Res (2021) 52:73
bounding to protein for 60 min at 4 ℃. Then the beads
were washed five times with buffer A [150 mM KCl,
25 mM Tris pH 7.4, 5 mM EDTA, 0.5 mM DTT, 0.5%
NP40, 1 mM PMSF, 100 U/mL SUPERAsin and 1 × pro-
tease inhibitor cocktail (Sigma)]. After rotating at 1000 g
for 5 min, the precipitate was added with the protein
loading buffer and loaded to precast 12% gradient Bis–
Tris gel for further analysis by Coomassie brilliant blue
staining. The different strips were then cut for mass spec-
trometry analysis.

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