Cloning, Prokaryotic Soluble Expression, and Analysis of Antiviral Activity of Two Novel Feline IFN-? Proteins

Wang, Xiaona; Li, Fengsai; Han, Meijing; Jia, Shuo; Wang, Li; Qiao, Xinyuan; Jiang, Yanping; Cui, Wen; Tang, Lijie; Li, Yijing; Xu, Yi-Gang

The fusion protein expressed by the pCold-feIFN-ω/BL21 bacteria cells was subjected to cleavage by the 3C protease (Sigma, St. Louis, MO, USA). The target recombinant feIFN-ω protein was then purified using Ni 2+ affinity chromatography columns according to the manufacturer's instructions, followed by confirmation analysis using SDS-PAGE. The purified feIFN-ω protein was stored at −80 • C until use. VSV was used as a virus model to evaluate the antiviral activity of the recombinant feIFN-ω via the in vitro microdose cytopathic effect inhibition assay (MCIA) according to the method described previously [12, 26] with slight modifications. Briefly, 100 µL of the purified feIFN-ω sample was serially diluted ten-fold in DMEM containing 10% FBS and transferred to confluent F81 cell monolayers in 96-well cell culture plates, and then incubated at 37 • C in 5% CO 2 for 18 h. F81 cells without IFN treatment were used as a control group. After incubation, VSV (MOI = 1) was prepared as described above, added into the 96-well plate, and incubated for 8-12 h until the CPE of the cells in the viral control group reached 100%. Next, the culture medium was removed, and the cells were stained with 0.2% crystal violet in 20% ethanol at 37 • C for 30 min. The cells were then destained with 0.1% acetic acid in 50% ethanol at 37 • C for 5 min before determining the absorbance of each well at 595 nm. Each sample was performed with eight biological replicates and three technical replicates. The antiviral activity of the IFN was calculated as the method previously described, which is expressed as units per milligram according to the ratio of IFN titer and protein concentration [27] . In parallel, the INTERCAT IFN antiviral drug (Toray Industries, Tokyo, Japan) was used as an IFN treatment control. In addition, we determined the species-specific antiviral activity of the recombinant feIFN-ω by conducting MCIAs using VSV in F81, Vero, MDCK, MDBK, and PK-15 cells. Broad-spectrum antiviral activity of the recombinant feIFN-ω was determined by conducting MCIAs using VSV, FCoV, CPV, BVDV, and PEDV.