Recombinant Lactobacillus casei Expressing Capsid Protein VP60 can Serve as Vaccine Against Rabbit Hemorrhagic Disease Virus in Rabbits

Wang, Li; Xia, Tian; Guo, Tiantian; Ru, Yi; Jiang, Yanping; Cui, Wen; Zhou, Han; Qiao, Xinyuan; Tang, Lijie; Xu, Yigang; Li, Yijing

The recombinant L. casei pPG-eGFP-VP60/LC393, pPG/LC393, and LC393 were cultured overnight in MRS broth and harvested by centrifugation at 9000 × g for 10 min at 4 °C After cell lysis and centrifugation, the supernatant was subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot assay. The proteins in the supernatant were separated by SDS-PAGE and electrotransferred onto PVDF membranes (Millipore, Milford, MA, USA). The immunoblot was then incubated with the following primary antibodies: Rabbit polyclonal anti-VP60(VP1) prepared in our laboratory (at the dilution of 1:200), and mouse monoclonal anti-eGFP (at the dilution of 1:5000) (Abcam, Cambridge, MA, USA) eat 4 °C overnight. Then, the immunoblot was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse IgG 1 : eGFP, cleaved with SacI and KpnI, was inserted into the corresponding sites of pMD18-T simple-VP60 digested by SacI and KpnI; 2 : eGFP-VP60 fragment, cleaved with SacI and ApaI, was inserted into constitutive expression vector pPG-T7g10-PPT; 3 : Cm resistance gene in pPG-T7g10-eGFP-VP60 (Cm + ) was disrupted by MunI and NcoI, and recombinant plasmid pPG-T7g10-eGFP-VP60 was obtained by blunt end ligation.