Elp il1b

The target sequence of porcine NLRP3 shRNA was 5′-GGTGACCTCATATGACTAA-3′. Annealed short hairpin RNA (shRNA)-synthesized cDNA fragments corresponding to the cDNAs of swine NLRP3 genes were digested with BamHI and EcoRI and ligated into the pLVX vector (HANBIO, Shanghai, China), which was named lentivirus expressing NLRP3 shRNA (shNLRP3). A non-effective shRNA cassette in the pGFP-C-shLenti shRNA vector plasmid was used as the negative control and purchased from ORIGENE (Rockville, MD, USA). The lenti vector encoding shNLRP3 was transfected into HEK293 cells together with psPAX2 and pMD2.G with Lipofectamine 2000 (invitrogen, Waltham, MA, USA). Culture supernatants were harvested at 48 and 72 h, then filtered with a 0.45 μm filter, and centrifuged at 72,000× g for 120 min at 4 °C. PK-15 cells were infected with supernatants containing lentiviral particles. The shRNA knockdown efficiency was assessed by Western blot analysis. The IL-1β level and IL-1β relative mRNA levels in infected PK-15 cells were measured by ELISA (ELISA kit from RayBiotech, Inc., Catalog #: ELP-IL1b) and qRT-PCR (List of primers are given in Table 1).

To detect the level of IL-1β in sera, organs, and cultured medium supernatants, ELISA kits were purchased from RayBiotech, Catalog #: ELP-IL1b (Peachtree Corners, GA, USA).