4.2. Expression of the VP8* Protein in E. coli and Antiserum Production

The prokaryotic expression vector pET32a-VP8* was constructed and transformed into E. coli Rosetta (BE3). The recombinant protein was expressed by induction with isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 1 mM when absorbance at 600 nm reached 0.5. After induction for 3 h at 37 °C, cells were harvested by centrifugation at 10,000× g for 15 min at 4 °C [49]. Proteins were separated on a 12% SDS-PAGE gel and transferred onto a PVDF membrane. The membrane was blocked with TBS containing 5% bovine serum albumin (BSA) at 37 °C for 2 h, washed five times with TBST, and incubated with mouse antirotavirus serum at 4 °C overnight. Following washing five times, the membrane was incubated with an HRP-conjugated goat anti-mouse IgG for 1 h at room temperature. The blot filters were visualized by the ECL Western Blot Detection Kit as specified by the manufacturer [50].

Porcine rotavirus vaccine was administered intraperitoneally to BALB/c mouse (20 g), followed by three boosts at 7-day intervals. Blood samples were collected on day 3 after the last injection. The antiserum was pooled together and stored at −80 °C [51]. The titers were determined by enzyme-linked-immunosorbent serologic assay (ELISA). All animal experiments were performed in accordance with the guidelines for the care and use of laboratory animals and approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University (approval number: DY2018203046).