Syncrip facilitates porcine parvovirus viral dna replication through the alternative splicing of ns1 mrna to promote ns2 mrna formation
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- Plasmid constructions and reactions
| Materials and methods
Cell culture and virus PK-15 and HEK293T cells were purchased from the American Type Culture Collection (ATCC, Manas- sas, VA, USA). These cells were cultured in Dulbecco modified eagle medium (DMEM) (Gebico, USA). All media were supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sijiqing, Hangzhou, China) in 5% CO 2 culture. PPV XY strain (Genbank: MK993540) was propagated in PK-15 cells. The crude PPV prepara- tion was purified using ultracentrifugation over sucrose cushions (2 mL of 50% sucrose plus 2 mL of 20% sucrose) by Optima XPN-100 Ultracentrifugation with a SW 41 Ti rotor, at 200 000 g for 2 h. Virus titers in the culture supernatants were determined by the Reed-Muench method [ 28 ]. Plasmid constructions and reactions PPV open reading frame 1 (ORF1) coding NS1 protein and three deletion mutant fragments were constructed using the overlap PCR approach from PPV genome and cloned into the pCI-neo vector. SYNCRIP protein was constructed from PK-15 cDNA and sub-cloned into the pCDNA3.0 vector. All sequences were confirmed by sequencing analysis (Sangon Biotech, Shanghai, China). For the construction of NS2 knockout Y-PPV clone (Y-PPV NS2− ), the three CTC in NS2 ORF of Y-PPV were mutated to TAG using the overlap PCR approach, as described previously [ 29 ]. CRISPR/Cas9 knockout cell: Lentiviral vector LentiC- RISPR v2 with puro resistance gene was used to clone sgRNA sequences between BsmB I sites. The sgRNA sequences were used to knockout SYNCRIP: SYN- CRIP-1: 5′-CTA TTG ACG TCT TCA TCA TAG GTA CC-3′; SYNCRIP-2: 5′-CAC CGA GAA TTC AAT GAA GACGG CGCA-3′. pLenti-NS2 and pLenti-SYNCRIP constructs: Lentivi- ral vector pCDH-EF1-MCS-T2A-Puro was used to clone optimized NS2 and SYNCRIP ORF between the restric- tion sites Nhe I and Not I. The PCR primers used in this study are shown in Table 1 . tải về 5.65 Mb. Chia sẻ với bạn bè của bạn:
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