Luminol chemiluminescence reagent kit

To confirm the successful rescue of recombinant virus rH120-QX(S), the P5 generation of the rescued virus was checked using Western blot. Briefly, 100 μl of allantoic fluid was lysed in a 5× loading buffer (Beyotime Biotechnology, China) and denatured for 10 min at 100°C. The 10 μl samples were resolved on a 10% SDS-PAGE and transferred to a 0.45 μm nitrocellulose membrane (Pall Corporation, USA). Membranes were blocked in 5% non-fat milk for 1 h, followed by incubation with IBV N polyclonal antibody diluted in blocking buffer overnight at 4°C. The membrane was then incubated with secondary antibody diluted in blocking buffer for 1 h at room temperature. Between and after the incubation, the membrane was washed three times with washing buffer (0.1% Tween in TBS). The signals were developed with a luminol chemiluminescence reagent kit (Share-bio, China) and detected using the Tanon 4600 Chemiluminescent Imaging System (Bio Tanon, China).
To further confirm the successful rescue of rH120-QX(S), the RNA was extracted from 500 μl of allantoic fluid. RT-PCR was performed with QX S gene primers and the S gene fragment was checked by agarose gel electrophoresis. Furthermore, the full-length genome was obtained by RT-PCR (Q5^® super fidelity PCR kit, New England Biolabs, USA) with primers in Supplementary Table 1 and sequenced (Genewiz, China).

Cells were lysed in 2x protein loading buffer (20 mM Tris-HCl, 2% SDS, 100 mM DTT, 20% glycerol, 0.016% bromophenol blue). Cell debris was pelleted at 15000xg for 10 min and 10 μg of the cleared cell lysates were resolved on a 10% SDS-PAGE and transferred to 0.45 μm nitrocellulose membrane (GE life Sciences). Membranes were blocked in blocking buffer (5% non-fat milk, TBS, 0.1% Tween 20) for 1 h, followed by incubation with primary antibody diluted in blocking buffer as indicated in S1 Table overnight at 4°C. The membranes were then incubated with secondary antibodies diluted in blocking buffer as indicated in S1 Table for 1 h at room temperature. Between and after the incubations, membranes were washed three time with washing buffer (0.1% Tween in TBS). The signals were developed with luminol chemiluminescence reagent kit (Share-bio) and detected using Tanon 4600 Chemiluminescent Imaging System (Bio Tanon).

H1299 or DF-1 cells were mock-infected or infected with IBV or rIBV-nsp15-H238A at 1 MOI for 20 h. Total RNA was extracted by using Trizol according to the manufacturer’s protocol. 2 μg RNA was spotted on Hybond-N+ membrane (GE Healthcare) and followed with UV crossed-linking (120 mJ/cm²) by using SCIENTZ 03-II (Scientz Biotech). After blocking with 5% non-fat milk dissolved in DEPC treated TBS, the membrane was incubated with mouse anti-dsRNA J2 antibody overnight at 4°C, followed by incubation with goat anti-mouse secondary antibody for 1 h. Between and after the incubations, membranes were washed three time with washing buffer (0.1% Tween in TBS). The dsRNA signals were developed with luminol chemiluminescence reagent kit (Share-bio) and detected using Tanon 4600 Chemiluminescent Imaging System (Bio Tanon).