Co-expressing VP2 Protein Of Porcine Parvovirus With E.coli Heat-labile Toxin B Subunit Protein In Recombinant Lactobacillus Casei And Immunology Evaluation

Lactobacillus casei ATCC 393 (L.casei 393) was selected as an antigen delivery vehicle for the development of live mucosal vaccine. The main protective antigen VP2 of porcine parvovirus and E.coli heat-labile toxin B subunit were selected as the target gene. We constructed recombinant L.casei 393 systems which co-expressing VP2 protein and E.coli heat-labile toxin B subunit protein. The immunogenicity responses induced by oral mucosal immunizations with recombinant strains which with LTB or without LTB were compared and two kinds of recombinant strains expressing interest protein in different cellular locations were also analyzed.We designed primers with Primer Premier 5.0 software according with target gene and considering the characters of fusion expression vector plasmid. About 1750bp gene fragment (VP2) and 375bp gene fragment were amplified by PCR using the plasmid pMD18-T-VP2 and the plasmid pMD18-T-LTB. The PCR products were digested by restriction enzyme, linked by T4 DNA ligase. About 2.1kbp gene fragment (VP2-LTB) was amplified by PCR using the product of linked. The interest gene fragment VP2-LTB was cloned into plasmid pMD18-T simple followed enzyme digestion, PCR identification and sequence analysis, and the recombinant plasmid was named pMD18-T-VP2-LTB. Then the pMD18-T-VP2-LTB plasmid was doubly digested by BamHI and XhoI, and the interest gene fragment VP2-LTB was obtained by agar gel purification, linked with expression vector pPG-1 and pPG-2 doubly digested by BamHI and XhoI, giving rise to pPG-1-VP2-LTB and pPG-2-VP2-LTB. The recombinant plasmids pPG-1-VP2-LTB and pPG-2-VP2-LTB were electroporated into L.casei 393 respectively, generating pPG-1-VP2-LTB/- L.casei 393 and pPG-2-VP2-LTB/L.casei 393 followed enzyme digestion, PCR identification and sequence analysis.The recombinant strains pPG-1-VP2-LTB/L.casei 393 and pPG-2-VP2-LTB/L.casei 393 constructed in this study were induced by 2% lactose in MRS medium to express recombinant interest protein. The recombinant strain pPG-1-VP2-LTB /L.casei 393 of cell surface expression was induced and about 80kD protein was detected with SDS-PAGE according with the theoretic molecule weight. The result of Western blot indicated that the expressed protein possessed the antigenic specificity which could be recognized by mouse anti-PPV serum. The indirect immunofluorescence and immunoelectron microscope test also showed that the expressed protein was displayed on the cell surface of L.casei 393. The recombinant strain pPG-2-VP2-LTB /L.casei 393 of secretion expression was induced by 2% lactose in MRS medium and about 75kD protein was detected via SDS-PAGE in the induced recombinant strain and culture supernatants according with the theoretic molecule weight. The result of Western–blot indicated that the expressed protein possessed the antigenic specificity same as the native virus protein. The experiment indicated that the interest protein was expressed and secreted into the culture supernatant.To identify whether the recombinant strains have the ability to induce systemic and mucosal antibody responses and the value of the LTB as mucosa adjuvant, five groups of ten female mice were immunized via oral route with pPG-1/L.casei 393, pPG-1-VP2/L.casei 393 pPG-1-VP2-LTB /L.casei 393, pPG-2-VP2/L.casei 393, pPG-2-VP2-LTB/L.casei 393 respectively. The immune pro- tocol was administered on three consecutive days at days 0, 1and 2. A booster immunization was given at days 14, 15 and 16 and a second booster was given at days 28, 29 and 30, the third boost- er was given at days 42, 43 and 44, the mice were fed with 109 recombinant strains. Serum of mice were collected before immunization and 7, 21, 35, 49 days after immunization. Fecal pellets were collected before immunization and 1, 5, 12, 19, 26, 33, 40, 47, 54 days after immunization. Ophth- almic wash and vaginal samples were obtained by washing the eyes and the vagina with 100μl phosphate-buffered saline (PBS) before immunization and 7, 21, 35, 49 days after immunization respectively. All samples were stored at–20℃until required. Finally, we determined the level of sIgA and IgG in those samples.To assess mucosal immune responses, specific IgA levels in mucosal samples were determined by ELISA. Specific IgA reached a high level in the fecal pellets,ophthalmic and vaginal wash. In contrast,only background levels of antibodies were detected in control animals. The IgA levels of mice administered with pPG-1-VP2-LTB/L.casei 393 or pPG-2-VP2-LTB/ L.casei 393 are higher than those administered with pPG-1-VP2/L.casei 393 or pPG-2-VP2/ L.casei 393 in fecal, ophthalmic and vaginal samples. This is due to co-expressing of mucosal immunoadjuvant LTB. This indicates that LTB can enhance the mucosal system response. This study has highlighted the potential of LTB as a safe and effective adjuvant.IgG titers of serum in mice given pPG-1-VP2-LTB/L.casei 393 or pPG-2-VP2-LTB/ L.casei 393 were higher than pPG-1-VP2/L.casei 393 or pPG-2-VP2/L.casei 393. It is prominence when compare with given pPG-1/L.casei 393 and the recombinant L.casei 393 of secretion expression type could elicit higher immune response level than that induced by cell surface expression type. In this study, oral administration of recombinant strains displaying VP2-LTB protein antigens induced both systemic and mucosal immune responses. Oral immunization elicited specific mucosal responses at the site of gastrointestinal tract, as well as the remote mucosal sites. And the sIgA titers in mice given pPG-1-VP2-LTB/L.casei 393 or pPG-2-VP2-LTB/L.casei 393 were higher than given pPG-1-VP2/L.casei 393 or pPG-2-VP2/L.casei 393. The immune exclusion and elimination of the pathogen at the mucosal surfaces by secretory IgA is crucial in preventing porcine parvovirus.The result of neutralization ability test showed that the neutralization ability of serum antibody was 1:304,1:317,1:338,1:352 which was induced by pPG-1-VP2/L.casei393,pPG-2-VP2/L.casei 393,pPG-1-VP2-LTB/L.casei 393,pPG-2-VP2-LTB/L.casei 393 respectively after 49d oral administration.This is the first report on the cloning and expression of recombinant LTB and PPV antigen in Lactobacillus. The results obtained so far demonstrate that lactobacillus are capable of delivering antigen to the mucosal and systemic immune systems following oral immunization. When VP2 was co-expressed with LTB, it showed much stronger mucosal immune responses. It indicated that LTB with qualified immunoreactivity, antigenicity and adjuvanticity could be used to develop genetically engineered vaccine. It should be suitably applicable as an immunoadjuvant for mucosal vaccines.All theses work established a good foundation for further study on the new and effective recombinant oral vaccine of porcine parvovirus.