Auxotrophic Expressing Porcine Rotavirus VP4 Constructed Using CRISPR-Cas9D10A System Induces Effective Immunity in Mice

Porcine rotavirus (PoRV) mainly causes acute diarrhea in piglets under eight weeks of age and has potentially high morbidity and mortality rates. As vaccine carriers for oral immunization, lactic acid bacteria (LAB) are an ideal strategy for blocking PoRV infections. However, the difficulty in knocking out specific genes, inserting foreign genes, and the residues of antibiotic selection markers are major challenges for the oral vaccination of LAB. In this study, the target gene, alanine racemase (), in the genome of strain W56 () was knocked out to construct an auxotrophic strain () using the CRISPR-Cas9D10A gene editing system. A recombinant strain (pPG-alr-VP4/) was constructed using an electrotransformed complementary plasmid. Expression of the alr-VP4 fusion protein from pPG-alr-VP4/ was detected using Western blotting. Mice orally immunized with pPG-alr-VP4/ exhibited high levels of serum IgG and mucosal secretory immunoglobulin A (SIgA), which exhibited neutralizing effects against PoRV. Cytokines levels in serum detected using ELISA, indicated that the recombinant strain induced an immune response dominated by Th2 cells. Our data suggest that pPG-alr-VP4/, an antibiotic-resistance-free LAB, provides a safer vaccine strategy against PoRV infection.