Anti-TGEV Miller Strain Infection Effect of Lactobacillus plantarum Supernatant Based on the JAK-STAT1 Signaling Pathway

Wang, Kai; Ran, Ling; Yan, Tao; Niu, Zheng; Kan, Zifei; Zhang, Yiling; Yang, Yang; Xie, Luyi; Huang, Shilei; Yu, Qiuhan; Wu, Di; Song, Zhenhui

IPEC-J2 cells were inoculated into 6-well plates at 1.8 × 10 6 /mL. The IPEC-J2 cells treated with Lp-1s for 1.5 h were exposed to TGEV (MOI = 0.1) for RNA extraction and protein sampling. When the cells reached 90% confluence, RNA was extracted and reverse transcribed into cDNA and quantified at 500 ng/mL. Absolute fluorescence quantitative PCR was performed using fluorescence quantitative PCR. The reaction parameters were as follows: Pre-denaturation at 95 • C for 3 min, followed by 40 cycles of denaturation at 94 • C for 30 s, annealing at 60 • C for 30 s, and prolongation at 72 • C for 30 s. The reaction for each sample was repeated three times. The Bio-Rad CFX Manager random matrix method was used to analyze the linear relationship between cycle threshold (CT) value and the copy number to calculate the copy number of the TGEV-N gene. Protein samples were also extracted at the same time point and detected using western blotting. The primary antibody was a mouse monoclonal antibody against TGEV-N, and the secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-mouse antibodies (Proteintech,Wuhan,China). The immunoreactive protein bands were visualized using a Vilber fusion FX5 chemiluminescent imager.