Immunogenicity Of The Truncated IBV S1 Protein Surface-Displyed On Lactobacillus Casei

The truncated infectious bronchitis virus gene S1 was expressed in Lactobacillus casei,and the chicken was immunized via a mucosal route to induce a systemic immune response and local mucosal immunity.The target gene fragment of IBV S1 was amplified by RT-PCR and the target fragment was ligated into the vector pQE-30.After the identification was correct,the protein was expressed and purified;The gene S1 fragment was constructed on the shuttle plasmid pLA and transformed into the E.coli expression system.After the identification was correct,the extracted plasmid was electro transformed into Lactobacillus casei.The resulting recombinant Lactobacillus casei strain was named pLA-S1/L.casei.Western blot was used to detect the expression of the target protein S1,and a fusion protein of about 60 kDa was obtained,which was in agreement with the expected results.The results of Western blot showed that protein S1 can react specifically with anti-IBV multi-antibody serum and has a very good reactivity.Indirect immunofluorescence showed that green fluorescence was observed in recombinant pLA-S1/L.casei under fluorescence microscope,indicating that the recombinant Lactobacillus casei expresses the exogenous protein of interest.The light scattering detector of flow cytometry could capture the fluorescence of recombinant Lactobacillus casei on the surface of the cell wall,and the expression rate of recombinant Lactobacillus casei expressed IBV S1 protein was 86.88%.We can detect the luminescence of the bacteria under a laser confocal microscopy.The bright field and green fluorescence of Merge shows that the luminescence occurs on the surface of the bacteria.Confocal laser scanning shows that the foreign protein can be displayed on the surface of Lactobacillus casei.In order to determine the immune pathway of recombinant pLA-S1/L.casei,the obtained recombinant Lactobacillus casei was administered orally and intranasally to the experiment group chicken,and set up the control group.Indirect ELISA was used to detect the oral pLA-S1/L.casei group.Serum IgG antibody levels in oral H120 live vaccine groups were significantly higher than those in other groups.Indirect ELISA was used to detect sIgA titer in small intestine washing fluid.Oral pLA-S1/L.casei and oral commercial live vaccines had significantly higher antibody titers than other groups.And oral recombinant pLA-S1/L.casei group can maintain relatively stable sIgA antibody levels.The experimental results show that the oral pLA-S1/L.casei group immune effect is better than the nasal eye drops group,so the best route of immunization in the recombinant pLA-S1/L.casei is oral immunization.In order to determine immunization times,twice and three times inoculation group were used as experiment groups and a control group was set up.The levels of serum IgG specific antibody and small intestine washing fluid sIgA was detected by indirect ELISA.The results showed that the IgG-specific antibody level and s IgA level in twice and three times inoculation group were significantly higher than the primary immunity level.There was no significant difference between twice and three times inoculation group.In the twice and three times inoculation group,the antibody level in 10~9 CFU/mL and 10~100 CFU/mL groups were significantly higher than 10~8CFU/mL group and the control group(P<0.01).And there was no significant difference between10~9 CFU/mL and 10~100 CFU/mL groups.The proliferation of spleen T lymphocytes was observed at day 7 post challenge.Data showed that the stimulation index of pLA-S1/L.casei group was significantly higher than that of other groups.The above results indicate,The results showed that IBV S1 protein was successfully displayed on the surface of Lactobacillus casei.The protective rate of the immunized group was comparable to that of the H120 immunized group,but the safety was high.