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 seeded at a density of 1.5 × 10 6 in cell slides in 24-well culture dishes. These cells were set as three experimental groups comprising an Lp-1s optimal concentration treatment group, a TGEV alone infection group, and a blank (uninfected) control group, when they reached 90% confluence. The cells were sampled at 12, 24, and 48 hpi; washed three times with PBS; fixed with 4% paraformaldehyde at 37 • C for 1.5 h; washed three times with PBS; permeated by 0.3% Triton-X-100 for 10 min; washed three times with PBS; and blocked by 1% bovine serum albumin for 30 min at room temperature. Primary antibodies comprising anti-p-STAT1 protein rabbit polyclonal antibodies and anti-TGEV-N mouse monoclonal antibodies were added and incubated overnight in 4 • C in a wet box. The cells were then washed three times with PBS, proportionally added CY3-goat anti-rabbit fluorescence and fluorescein isothiocyanate (FITC)-labeled goat anti-mouse fluorescent secondary antibodies were then added, the cells were incubated for 1 h in a dark room at 37 • C, incubated with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) for 5 min, rinsed with PBS. Laser confocal microscopy was then used to observe p-STAT1, its nuclear translocation, and the TGEV N protein. The protein levels were analyzed using the ZEN software.