| Porcine rotaviruses are classified in the genus Rotavirus, of the family Reoviridae. Porcine rotaviruses are the major cause of acute diarrhea in the piglets. Rotaviruses are transmitted via the fecal-oral route. According to the feature of mucosa infection, effective vaccine which can induce local and systematic immunity is of great significance for the prevention and cure. LTB is the B subunit of Escherichia coli( E. coli) heat-labile ,which is nontoxic and can be co-immuned with many proteantigen or nonprotein antigen by different immunity ways.LTB can strengthen mucosa and systematic immune response,which can eliminate immunity tolerance and induce permanent immunity memory.In the study, Lactobacillus casei ATCC 393 acted as antigen delivery carrier for oral vaccination according to the feature of porcine rotavirus infection and immunity.In addition, lactobacillus has many beneficial effects to the intestinal tract. It has adjuvant effect and the feature of non-specificial anti-infection. The major protective antigen of porcine rotavirus (VP4) and VP4-LTB were used as the antigen model and inserted into secretion expression plasmid. The recombinant Lactobacillus expressing VP4 and VP4 with LTB protein were constructed and the immunogenicity of the recombinant strains were compared and analyzed.We designed primers P1 and P2 with oligo6.0 software according with target gene and considering the characters of fusion expression vector plasmid. About 756bp gene fragment (VP4) was amplified by PCR using the plasmid VP4-PGEX-6P-1 and linked to pMD18-T simple vector .Then it was digested by restriction enzyme,PCR and sequencing. The recombinant plasmid was digested by BamHI and XhoI.VP4 gene was obtained and linked with pPG-2 expression vector,which is electrotransformed into L.casei 393.We obtained recombinant plasmid named pPG-2-VP4 by restriction enzyme,PCR and sequencing.The recombinant Lactobacillus casei was named pPG-2-VP4/L.casei393.At the same time, About 375bp gene fragment ( LTB) was amplified by PCR using the plasmid pMD18-T-LTB with P3 and P4 by the same way. The recombinant plasmid was digested by SalI and XhoI.LTB obtained was linked with pMD18-T-VP4 digested by XhoI. VP4-LTB was amplified with P1 and P4 and linked with pMD18-T simple vector. We obtained recombinant plasmid named pMD18-T-VP4-LTB by restriction enzyme,PCR and sequencing. The recombinant plasmid was digested by BamHI and XhoI.VP4 gene was obtained and linked with pPG-2 expression vector,which is electrotransformed into L.casei 393. We obtained recombinant plasmid named pPG-2-VP4-LTB by identification.The recombinant Lactobacillus casei was named pPG-2-VP4-LTB/L.casei393.The recombinant strains constructed in this study were induced by two percent of lactose in MRS medium to express interest protein. The lysates of the cells were analyzed by SDS-PAGE. Coomassie blue gel staining showed that 27kDa and 40KD fusion protein were expressed in lysates,which is corresponding to the theoretical value . The localization of the VP4 and VP4-LTB protein lactose-induced were analyzed via Western blotting .Immunoreactive bands (27KD,40KD) were detected. The immunofluorescence was developed with the mouse anti-VP4 serum. The results of the immunofluorescence indicated that there was green-yellow fluorescence on the cell surface of the recombinant strains. To identify the value of recombinant strains as live organism vaccine and the effect of LTB as mucosal immunoadjuvant, immunology evaluation was carried out with BALB/c mice as experimental animals.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. Booster immunization was every two weeks and once a day. Three immunity were administered. At the same time , the mice immunized with pPG-2 as negative control. Specific sIgA levels in the fecal pellets ,milk, vaginal and ophthalmic wash and IgG levels in the serum were determined. Specific IgA reached a high level in the the fecal pellets ,milk, vaginal and ophthalmic wash by ELISA. In contrast, the titers of antibodies and the duration in mice given the recombinant strain expressing VP4-LTB are more significant than the mice given the recombinant strain expressing VP4,but the titers of IgG in the serum are similar and in the high level. Splenocytes proliferation and determination of IL-4and IFN-r indicated that immunization of recombinant strains produced significant cellullar immunologic response.Neutralization ability of the induced antibodies were determined by virus neutralization test. The result of neutralization ability test showed that the neutralization ability of serum antibody in mice administered with pPG-2-VP4/L.casei393 was 1: 99 and the neutralization ability of serum antibody in mice administered with pPG-2-VP4-LTB /L.casei393 was 1: 199. Recombinant strains as oral vaccine gained significant immunity effect. The result indicated that recombinant Lactobacillus casei expression system as oral vaccine has potential value and provide the base for studying the vaccine of porcine rotavirus. |
