Construction Of The Recombinant Lactococcus Lactis Expressing GPV VP3 And CPV VP2 Structural Proteins

Both Canine parvovirus (CPV) infection and Goose parvovirus (GPV) infection (known as Gosling Plague) are highly contagious and lethal sickness, the former with the most typical lesions of hemorrhagic enteritis and strikingly decreasing of white blood cell and acute myocarditis in dogs, and the latter is characteristic of embolism of intestinal tract in goslings and muscovy ducklings under the age of 5 weeks. Because the attenuated vaccines for preventing and controling the diseases described above have their own inevitable shortcomings and can not achieve the desired effect of protection against the parvovirus infection. Therefore, to develop much more safe and effective vaccines have been targeted urgently.Bacterial vector-based vaccine is constructed by inserting the DNA fragments which encode some specific antigens from the pathogen or the target antigens into the attenuated pathogens or symbiotic bacteria as live vehicles, to express and present the encoded antigen and effectively stimulate immune response of the host cells as the attenuated pathogens or symbiotic bacteria colonize, multiplicate and grow in the host. Bacterial vector-based vaccine with capability of producing both humoral and cellular immunue responses, especially mucosal immunue response can prevent one or multiple specific pathgen infection as we expect to. It is of note that the attenuated pathogens as carrier have a certain risk of virulence revert, generally regarded as safe (GRAS) organisms as vehicle have been studied and developed. Among them, the non-pathogenic Gram-positive lactic acid bacteria (LAB) are typical representative. LAB is the probiotics in human and animal intestine and has a variety of properties which make them attractive candidates for oral vaccination purposes, e.g. adjuvant properties, mucosal adhesive properties, low intrinsic immunogenicity and immunomodulation capacity.As we know, both GPV VP3 and CPV VP2 capsid protein could induce the neutralizing antibodies and play a principal immunoprotection role. For constructing the recombinant Lactococcus lactis as live vehicles expressing the both target proteins, the GPV VP3 gene and CPV VP2 gene were amplified by PCR and the templates of the nucleic acid of GPV infected 13-day-old goose embryo allantoic fluids and the recombinant plasmid DNA pGEX-6P-1-VP2 carrying CPV VP2 gene, respectively, and cloned into pMD18-T simple vector. After the positive clones being verified by analysising with restriction enzymes and the correct sequences of the GPV VP3 and CPV VP2 genes being sequened, the GPV VP3 gene and CPV VP2 gene were cloned into the Lactococcus lactis-based vectors pNZ3004 and pXYSEC:Nuc, respectively, and the recombinant vectors pNZ3004-GVP3 and pXYSEC:CVP2 confirmed by the methods of combination of PCR and restriction enzymes analysis were transformed into Lactococcus lactis NCDO2118 by electroporation, resulting in the recombinant Lactococcus lactis strains carrying pNZ3004-GVP3 or pXYSEC:CVP2. The recombinant GPV VP3 and CPV VP2 capsid protein extracted from the recombinant Lactococcus lactis cell lysate after inducible expression were detected by Western blot. The both expressed proteins could be recognized with responding goose sera angaist GPV and mouse sera against CPV, respectively. The optimal inducible expression condition is following: anaerobicly culturing at 30℃until it reach a suitable OD600(0.4～0.6), adding final concentration of 1% latose and 0.5% xylose for remaining 10 hours culture (about OD600 1.5), respectively. Based on the the recombinant Lactococcus lactis strains pNZ3004-GVP3/NCDO2118 and pXYSEC:CVP2/NCDO2118 expressing the GPV VP3 and CPV VP2 capsid protein, It may be promising to develop much more safe and effective vaccines of the recombinant Lactococcus lactis as a live vehicle against the parvovirus of GPV and CPV infections.