Construction Of Recombinant Lactococcus Lactis Expressing Neutraliaing Antigenic Domain Of Procine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae, was an intestinal pathogen of infectious disease of porcine which characterized by severe enteritis with anorexia, vomiting, acute diarrhea dehydration and significant mortality in swine. Histologically, the viruse cause destruction of villous enterocytes and villous atrophy within the jejunum and ileum. Morbidity and mortality in infected neonatal piglets less than 5 days old approach 100% because of severe diarrhea and dehydration. Porcine epidemic diarrhea has been widely distributed in the world, especially in the Eastern Asia and causing pig producers serious economical loss.Lactic acid bacteria (LAB), considered to be safe bacteria with a GRAS (generally regarded as safe) status, possesses many properties that has been thought to be an ideal vaccine which has a good perspective. An oral vaccine of expressing the antigen then induced efficient systemic and mucosal immune responses is very useful resulting in the inhibition of the virus.Lactococcus lactis NZ9000 was selected as an antigen delivery vehicle for the development of live mucosal vaccine. The neutralizing fragment of the spike protein ps420 was selected as the target gene. We constructed recombinant Lactococcus lactis NZ9000 systems expressing ps420. About 420bp gene fragment (ps420) was amplified by PCR using the plasmid pPG612.1-ps420. The products were linked with pMD-18-T Simple vector and digested by restriction enzyme. The target gene was digested by restriction enzyme, linked with expression vector pNZ8112 digested by restriction enzyme, giving rise to pNZ8112-ps420. The recombinant plasmids pNZ8112-ps420 was electroporated into Lactococcus lactis NZ9000 respectively, generating pNZ8112-ps420/NZ9000 followed enzyme digestion, PCR identification and sequence analysis.The recombinant strains pNZ8112-ps420 constructed was induced by Nisin in GM17 medium to express interest protein. The expression and localization of the protein from recombinant strains were detected via SDS-PAGE, Western blotting and indirect immunofluorescence. The lysates of the cells were analyzed by SDS-PAGE. Coomassie blue gel staining showed that 15KD fusion protein was expressed in lysates of pNZ8112-ps420. The localization of the ps420 protein nisin-induced was analyzed via Western blotting. Immunoreactive band (15KD) was detected, and corresponding immunoreactive bands did not display when they were not induced.Rabbits were immunized via oral route with pNZ8112-ps420/NZ9000 to identify whether the recombinant strain have the ability to induce systemic antibody responses. Each rabbit received dose of 1010 colony-forming units (c.f.u.)/mL of recombinant strains. The immune protocol was administered on three consecutive days at days 1,2 and 3. A booster immunization was given at days 15,16 and 17 and a second booster was given at days 29,30 and 31. Serum of rabbits were collected 7 days after every immunization and the results of neutralization test showed that the IgG in serum reach a comparative high level. The gene encoding PEDV ps420 protein could be certificated as a neutralizing antigen domain. All theses work established a good foundation for further study on the new and effective recombinant oral vaccine of porcine epidemic diarrhea.