Protease k

Before the DNA isolation, samples were treated with 2000 U/ml DNase I (New England BioLabs, Frankfurt, Germany) for 2 h at 37 °C to remove possible nucleic acid contaminants. After treatment, the enzymes were heated to the condition of inactivation at 75 °C for 10 min (this step reaction was only used to verify the distribution of serum DNA). First, total DNA was extracted from EVs by using DNA lysis buffer [0.5 % SDS, 0.05 M EDTA, 0.01 M Tris-HCl pH 8.0, 0.1 M NaCl, 200 μg/ml Protease K (Amresco, Solon, Ohio, USA)]. A total of 400 μl DNA lysis buffer was added to each tube of EVs. After mixing, the samples were incubated for 24 h at 55 °C. The serum that EVs had been extracted out of did not need the process above. Second, deproteinization was performed using a balance of phenol and chloroform. Third, DNA was precipitated using 3 M CH₃COONa, glycogen and absolute ethanol for 24 h at −20 °C and resuspended DNA with TE (0.001 M EDTA, 0.01 M Tris-HCl pH 8.0) at 37 °C for at least 16 h. The EVs DNA was quantified on a Nano Drop ND-2000 Spectrophotometer (Thermo Fisher Scientific).

As described previously [7 (link)], lysis buffer containing 0.5 mg/mL protease K (Amresco, US), 50 mM KCl (Sigma), 10 mM Tris-Cl, pH 8.0 (Invitrogen), 2.5 mM MgCl₂ (Sigma), 0.45% IGEPAL (Sigma), 0.45% Tween-20 (Promega, US), and DEPC-treated water (Promega) was used for the total RNA extraction. Briefly, 200 µL of lysis buffer was added to each well of 96-well plates that contained PIEs and was incubated for 30 min at 37 °C. Then, 100 µL of the PIE–lysis buffer mixture was transferred to a 96-well PCR plate, followed by incubation in a PCR cycler with the following parameters: 30 min at 65 °C and 2 min at 98 °C, and the samples were then cooled down to 4 °C. The RNA concentration was measured by NanoDrop2000c and adjusted to the same level. A Qiagen one-step RT-PCR kit was used for RVC detection using the primers, probe, and protocol described previously [18 (link)]. The Ct values from the RT-PCR were converted to FFU/mL based on a standard curve generated using the infectious titer of the cell culture-adapted RVC Cowden. Our calculations demonstrated that the mRNA quantities correlated with the CCIF (infectious virus titers) results (R² = 0.98).

To analyze the effect of different pHs (range 3.0–10.0) on the antifungal activity of rF2, the pH of the culture filtrate was altered using 2 mol/l NaOH or HCl and incubated for 2 h at 37 °C. Protein rF2 was exposed to a range of temperatures (40, 60, 80, 100, and 121 °C) for 30 min to study its thermal stability. To detect its stability under protease treatment, rF2 was digested using 1 mg/ml protease K, trypsin, and pepsin (Amresco, Radnor, PA, USA) for 60 min at 37 °C, respectively. The antifungal activities of the rF2 culture filtrates treated as detailed above were determined according to the method described by Zhao et al. [37 (link)]. The formula reported by Wong et al. was used to calculate the relative activity of the treated rF2 protein [38 (link)]. Three replicates for each treatment were assessed in three repeated experiments.