| Porcine transmissible gastroenteritis virus(TGEV) and porcine epidemic diarrhea virus(PEDV) are main pathogens that resulted in the pig acute enteritis and diarrhea. The mortality of infected piglets can reach 100%, which bring huge losses in the hog industry. 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.In the study, the major protective antigen of porcine transmissible gastroenteritis virus(6Ds) and porcine epidemic diarrhea virus(ps420) were used as the antigens model and inserted into constitutive Lactobacillus expression vector(p PG-T7g10-PPT), the recominant plasmid co-expressing 6Ds and ps420 protein were constructed which named p PG-T7g10-6Ds-ps420. The recominant plasmid was transformed into the Lactobacllus casei by electrotransformation to generate the recombinant bacteria named p PG-T7g10-6Ds-ps420/L.casei 393. The expressed proteins of recombinant strains were cultured for 16 h in in MRS broth 37 ℃ static and analyzed by Western-blot, the results of Western blot analysis indicated 80 kDa which are the special band as we expected. And on this basis we used the enhanced green fluorescent prote in(EGFP) gene as the reporter gene to reconstruct recombinant lactobacillus casei plasmid p PG-T7g10-EGFP-6Ds-ps420. We use the plasmid p PG-T7g10-EGFP-6Ds-ps420 connecting the EGFP, and they were transformed into the host strain Lactobacllus casei by electrotransformation to generate the recombinant bacteria named p PG-T7g10-EGFP-6Ds-ps420/L.casei 393. By the analysis of Western blot and confocal laser experiments, it showed that objective protein was expressed in the recombinant bacteria. The recominant plasmid p PG-T7g10-FEGFP-6Ds-ps420 chloramphenicol resistance marker was removed by restriction enzyme digestion, and transformed into lactobacillus casei by electricity transformation after blunt and ligation.The recombinant strains(free of antibiotic screening marker) with EGFP fluorescence signal were collected by flow cytometry and spread on the MRS culture, and the recombinant bacteria named p PG-T7g10-FEGFP-6Ds-ps420/L. casei 393. The recombinant bacteria p PG-T7g10-FEGFP-6Ds-ps420/L.casei 393 was analyzed by Western-blot, the results show that indicated 80 kDa which are the special band as we expected.To identify the value of recombinant strain(p PG-T7g10-FEGFP-6Ds-ps420/L.casei 393) as live organism vaccine, immunology evaluation was carried out with B ALB/c mice as experimental animals. BALB/c mice were divided into four groups at randam: PPG-T7g10-6Ds-ps420/L.casei 393 and p PG-T7g10-FEGFP-6Ds-ps420/L.casei 393, group PBS and pPG-T7g10-PPT/L.casei 393 were the negative control which inoculated nothing; the oral groups immunized with live vaccine 2×109CFU each mouse every two weeks and immunized continuing 3 days. Collecting serum levels, faeces and external genital tract rinses mucus samples at different time points to TGEV and PEDV whole virus as coating antigen to detect antibody levels in immunized samples by indirect ELISA method. The results show that p PG-T7g10-6Ds-ps420/L.casei 393 and pPG-T7g10-FEGFP-6Ds-ps420/L.casei 393 immunized groups Ig A and Ig G antibodies were significantly higher than PBS and p PG-T7g10-PPT/L.casei 393 group, pPG-T7g10-6Ds-ps420/L. casei 393 and p PG-T7g10-FEGFP-6Ds-ps420/L.casei 393 immune antibody level difference between groups was not significant. Splenic lymphocyte proliferation assay was measured by ELISA. The result indicated that 5μg/m L TGEV 6Ds or PEDV ps420 have stronger challenge than 5 μg/mL and 25μg/m L TGEV 6Ds or PEDV ps420. Neutralization ability of the induce antibodies were determined by virus neutralization test. The result of neutralization ability test sho wed that the neutralization ability to TGEV of serum antibody in mice administered with p PG-T7g10-6Dsps420/L. casei 393 was 1:100 and neutralization ability of serum antibody in mice administered with pPG-T7g10-FEGFP-6Ds-ps420/L. casei 393 was 1:63.8. The result of neutralization ability test showed that the neutralization ability to PEDV of serum antibody in mice administered with pPG-T7g10-6Ds-ps420/L. casei 393 was 1:112.2 and neutralization ability of serum antibody in mice administered with p PG-T7g10-FEGFP-6Ds-ps420/L. casei 393 was 1:100. No significant difference between groups.In conclusion, this study constructed recombinant lactobacillus casei pPG-T7g10-FEGFP-6Ds-ps420/L. casei 393 without antibiotic resistance gene which can express TGEV 6Ds and PEDV ps420 fusion protein. By animal immunization experiments confirmed that the recombinant strain has good immunogenicity, it laid the foundation for the further exploration of new TGEV and PEDV developed bivaalent oral vaccine. |
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