Construction Of Recombinant Lactobacillus Expressing VP4 Protein Of Porcine Rotavirus And Co-Expressing VP4 With LTB And Immunology Evaluation

Porcine rotaviruses are nonenveloped, segmented, doublestranded RNA viruses classified in the family Reoviridae. Porcine rotaviruses are the major cause of diarrhea in the piglets .Porcine rotavirus has been widely distributed in the world and cost pig producers serious loss.In this study, Lactococcus lactis NZ9000 and Lactobacillus casei ATCC 393 (L.casei 393) were selected as an antigen delivery vehicle for the development of live mucosal vaccine. The main protective antigen VP4 was selected as the target gene and E.coli heat-labile toxin B subunit protein was inserted. We constructed recombinant Lactococcus lactis NZ9000 and recombinant L.casei 393 systems expressing VP4 and co-expressing VP4-LTB.The immunogenicity responses induced by oral mucosal immunizations with recombinant strains were compared.We designed primers with oligo6.0 software according with target gene and considering the characters of fusion expression vector plasmid. About 756bp gene fragment (VP4) and 375bp gene fragment were amplified by PCR using the plasmid VP4-pGEX-6P-1 and the plasmid pMD-18T-LTB. The PCR products were digested by restriction enzyme, linked with expression vector pNZ8112 digested by restriction enzyme , giving rise to pNZ8112-VP4 and pNZ8112-VP4-LTB.The recombinant plasmids pNZ8112-VP4 and pNZ8112-VP4-LTB were electroporated into Lactococcus lactis NZ9000 respectively, generating pNZ8112-VP4/ NZ9000 and pNZ8112-VP4-LTB/ NZ9000 followed enzyme digestion, PCR identification and sequence analysis.At the same time, we obtained pPG-1-VP4/ L.casei 393 and pPG-1-VP4-LTB/ L.casei 393 by the same way.They also followed enzyme digestion, PCR identification and sequence analysis.The recombinant strains pNZ8112-VP4 and pNZ8112-VP4-LTB constructed were 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 immunofluorescence. The lysates of the cells were analyzed by SDS-PAGE. Coomassie blue gel staining showed that 27KD and 40KD fusion protein were expressed in lysates of pNZ8112-VP4 and pNZ8112-VP4-LTB, but not expressed when the same cells were grown in GM17 medium without Nisin . The localization of the VP4 and VP4-LTB protein nisin-induced were analyzed via Western blotting .Immunoreactive bands (27KD,40KD) were detected , and corresponding immunoreactive bands did not display when they were not induced. These results show that Nisin promoter from Lactococcus lactis NZ9000 could efficiently induce the expression of heterologous protein. The immunofluorescence was developed with the mouse anti-VP4 serum. The results indicated that there was green-yellow fluorescence on the cell surface of pNZ8112-VP4 and pNZ8112-VP4-LTB when induced. The cells uninduced did not display an immunofluorescence reaction and were dyed red by Evans blue.The recombinant strains pPG-1-VP4 and pPG-1-VP4-LTB constructed in this study were induced by lactose in MRS medium to express interest protein. The expression and localization of the protein from pPG-1-VP4 and pPG-1-VP4-LTB were also detected via SDS-PAGE, Western blotting, and immunofluorescence. The lysates of the cells were analyzed by SDS-PAGE. Coomassie blue gel staining showed that 40KD and 53KD fusion protein were expressed in lysates of pPG-1-VP4 and pPG-1-VP4-LTB, but not expressed when the same cells were grown in MRS medium without lactose . The localization of the VP4 and VP4-LTB protein lactose-induced were analyzed via Western blotting .Immunoreactive bands (40KD,53KD) were detected , and corresponding immunoreactive bands did not display when they were not induced. These results show that lactose promoter from L.casei 393 could efficiently induce the expression of heterologous protein. The immunofluorescence was developed with the mouse anti-VP4 serum. The results indicated that there was green-yellow fluorescence on the cell surface of pPG-1-VP4 and pPG-1-VP4-LTB when induced. The cells uninduced did not display an immunofluorescence reaction and were dyed red by Evans blue.To identify whether the recombinant strains have the ability to induce systemic and mucosal antibody responses, six groups of ten female mice were immunized via oral route with pPG-1, pPG-1-VP4,pPG-1-VP4-LTB,pNZ8112,pNZ8112-VP4 and pNZ8112-VP4-LTB respectively. Every mouse received dose of 109 colony-forming units (c.f.u.)/mL of recombinant strains, respectively. The immune protocol was administered on three consecutive days at days 1,2 and 3. A booster immunization was given at days 17,18 and 19 and a second booster was given at days 33,34 and 35. Serum of mice were collected 7 days after every immunization. Fecal pellets were collected 1, 2, and 7 days after every immunization. Ophthalmic wash were obtained by washing the eyes with 50μL phosphate-buffered saline (PBS) 7 days after every immunization. Vaginal samples were collected by washing the vagina with 200μL PBS 7 days after every immunization . Milk were collected 3,5,7 days after delivery. All samples were stored at–20℃until required.To maintain homeostasis in the mucosae,different defense mechanisms are involved in permanent and effective surveillance. One of these mechanisms is the secretory immune system through IgA antibodies. IgA prevents the invasion and colonization of pathogenic microorganisms and competes for receptors and metabolic substrates. 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-VP4 are higher than those administered with pNZ8112-VP4. The IgA levels of mice administered with pPG-1-VP4-LTB are higher than those administered with pNZ8112-VP4-LTB. This is because L.casei 393 has better colonization ability in intestinal tracts than Lactococcus lactis NZ9000. The IgA levels of mice administered with pPG-1-VP4-LTB or pNZ8112-VP4-LTB are higher than those administered with pPG-1-VP4 or pNZ8112-VP4 in fecal, ophthalmic and vaginal samples. This is due to the addition of mucosal immunoadjuvant LTB. This indicates that LTB can enhance the mucosal system response. Vaccines with low immunogenicity in particular require the use of a strong adjuvant to enhance the antigen-presenting .This study has highlighted the potential of LTB as a safe and effective adjuvant.IgG titers of serum in mice given pPG-1-VP4 or pPG-1-VP4-LTB were similar but higher than the mice given pPG-1. IgG titers of serum in mice given pNZ8112-VP4 or pNZ8112-VP4-LTB were similar but higher than the mice given pNZ8112. In this study, oral administration of recombinant strains displaying VP4 or VP4-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. The IgA antibodies can bind the antigen and minimize its entry with a consequent reduction in inflammatory reactions, which prevents a potentially harmful effect on the tissue . The immune exclusion and elimination of the pathogen at the mucosal surfaces by secretory IgA is crucial in preventing porcine rotavirus .Freshly isolated mouse splenocytes were seeded at 5×106 cells/mL and incubated for 66 h. 0.5μg/mL,5μg/mL and 25μg/mL VP4 were added to challenge.Cell culture supernatants were harvested and analyzed for the presence of cytokines. Splenic lymphocyte proliferation assay was measured by ELISA. The result indicated that 0.5μg/mL and 5μg/mL VP4 have stronger challenge than 25μg/mL VP4. Interleukin-4 (IL-4) and gamma interferon (IFN-γ) cytokine assays were performed using Biosource ELISA kits according to the manufacturer's instructions. The result indicated that all immunized groups secreted significantly more IL-4 and IFN-γthan the control group.Significant differences were seen among the groups. Immunized mice secreted increased levels of IL-4 that were significantly higher than IFN-γ. Overall, these data show that immunization of recombinant strains via oral route produced significant innate-type cytokine responses. It suggested that enhanced protection against infection of rotavirus .Furthermore, we collected the milk of mice 3,5,7 days after delivery.Specific IgA in the milk were detected by ELISA.The result indicated that there were high levels of specific IgA in the samples.It shows that antibodies produced by administration with recombinant strains can serve passive immunoprotection to the immature animals.In conclusion , the results obtained so far demonstrate that Lactococcus lactis and lactobacillus are capable of delivering antigen to the mucosal and systemic immune systems following oral immunization. Lactic acid bacteria as vaccine delivery vehicles are able to colonize different mucosal sites such as gastrointestinal tracts, genital tracts and could thus be used specifically to vaccinate against pathogens at their site of entry. Their harmLess nature make them more appropriate as an oral vaccine carrier to deliver heterologous antigens. The recombinant strains have better immunogenicity and can elicit stronger specific mucosal immune responses and systematic immune responses. When VP4 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 rotavirus.