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

In this study, the genes encoding feIFN-ω that were isolated by RT-PCR were subcloned as a KpnI and BamHI-generated (New England Biolabs, MA, USA) gene fragment into the prokaryotic soluble expression plasmid pCold-TF, giving rise to recombinant pCold-feIFN-ω. After that, the recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells and validated by PCR and sequencing analyses, thus generating the recombinant E. coli strain pCold-feIFN-ω/BL21. We then used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis to determine the optimal conditions for expression of the recombinant feIFN-ω in pCold-feIFN-ω/BL21 cells via induction by isopropyl β-D-thiogalactoside (IPTG, Sigma, St. Louis, MO, USA). Briefly, for optimization of the IPTG concentration, the recombinant E. coli strain was grown in Luria-Bertani broth (Sigma, St. Louis, MO, USA) supplemented with 100 µg/mL ampicillin at 37 • C until the optical density at 600 nm was approximately 0.5. Then, IPTG was added at final concentrations of 0.4 mmol/L, 0.6 mmol/L, 0.8 mmol/L, 1.0 mmol/L, or 1.2 mmol/L, and the cultures were continually cultivated for Viruses 2020, 12, 335 4 of 16 another 6 h. After centrifugation at 12,000× g for 10 min, the cell pellets were lysed and analyzed by 12% SDS-PAGE. To determine the optimal induction time, the recombinant strain was induced by the optimized final concentration of IPTG for 4 h, 6 h, 8 h, 10 h, and 12 h. Following centrifugation and cells lysis, the proteins were again analyzed by 12% SDS-PAGE.