Abstract:

The aim of this study was to construct a recombinant secreted Lactobacillus oral live vector strain co-expressing goose parvovirus (GPV) VP3 protein and goose interleukin-2 (gIL-2) protein. The VP3 gene was ligated with gIL-2 by enzyme digestion, and the signal peptide (SP) gene from Lactobacillus brevis 1.2028 was located on the N-terminal of fusion protein. The three genes (as a complete open reading frame) were subcloned into Lactobacillus integrated expression vector pMJ67, and then the recombinant plasmid pMJ-SP-GPVVP3-gIL2 was electro-transformed into Lactobacillus casei CECT5276 to construct the oral live vector vaccine strain. The recombinant Lactobacillus was identified by PCR method and immunogenicity of expression protein was tested by SDS-PAGE and Western blot methods. The specific anti-GPV antibody in serum, secretive immunoglobin A (sIgA) and spleen lymphocyte proliferation were detected to evaluate the immune effects after oral immunization goose with the recombinant Lactobacillus. The results showed that the recombinant plasmid has been constructed successfully, PCR proved that the plasmid pMJ-GPVVP3-gIL2 has been integrated into Lactobacillus genome, SDS-PAGE and Western blot could detect the GPV VP3-gIL2 protein band in supernatant and it could be recognized by the positive serum against GPV. Spleen lymphocyte proliferation responses results indicated that recombinant Lactobacillus could also induce an obvious cellular immune response, the lymphocytes proliferation level of GPV VP3-gIL2 group was obviously higher than GPV VP3 group and gIL2 group in the 4^th, 5^th and 6^th week. Neutralization antibodies could be produced in GPV VP3-gIL2 group, GPV VP3 group and gIL2 group, but the neutralization antibodies level of GPV VP3-gIL2 group was significantly higher than that of the latter two groups. Since the 2^nd week, the number of sIgA in intestinal mucosal fluid was significantly higher than GPV VP3 group and Vaccine group. Challenge experiment results showed that goose immunized with the GPV VP3-gIL2 fusion protein obtained 85% protection; the results confirmed that the recombinant Lactobacillus could enhance the protection against GPV. A recombinant oral live vaccine Lactobacillus CECT5276 (pMJ-GPVVP3-gIL2) secretive expressing fusion protein GPV VP3-gIL2 was constructed successfully, this recombinant Lactobacillus could be a basis to oral live vector vaccine for GPV infection.