Construction And Immunogenicity Of The Recombinant Lactobacillus Casei Coexpressing The Truncated F Protein Of NDV And S1 Protein Of IBV

To obtain NDV F and IBV S1 recombinant proteins for the establishment of a subsequent indirect ELISA method.According to the NDV F gene sequence and the IBV S1 gene sequence,specific primers were designed and synthesized,and the target fragment was amplified to construct the prokaryotic expression system expressing NDV F protein and IBV S1 protein.Western blot results showed that the recombinant protein has good reactivity and can be used in subsequent experiments.The recombinant pLA-F-S1/Lactobacillus casei(L.casei)expressing the truncated F protein and S1 protein were constructed for evaluating its immune efficacy.In this experiment,NDV F and IBV S1 partial epitopes were ligated in tandem with the shuttle plasmid PLA.Convert it to E.coli BL21(DE3).To filter positive recombinant plasmid,and electroporate it into L.casei,and obtain pLA-F-S1/L.casei.The positive strain was identified by PCR,and the recombinant protein was detected by SDS-PAGE and obtain a recombinant protein with a size of about 78 kDa,which was consistent with the expected result.Western blot results showed that the recombinant protein could bind to anti-NDV and anti-IBV whole virus serum and had good reactivity.The positive expression bacteria were screened by indirect immunofluorescence and analyzed by flow cytometry.The results showed that 94.10% of the recombinant bacteria expressed the specific protein.Laser confocal detection showed that the protein was successfully displayed on the surface of the recombinant bacteria.The 14-day-old chicks were selected for the test.The immunological methods of each group were oral + nasal drops.Twice immunized groups of pLA-F-S1/L.casei,three times immunized of pLA-F-S1/L.casei,pLA-F/L.casei group,pLA-S1/L.casei group,commercial vaccine group,pLA/L.casei group and PBS group were established.Indirect ELISA was used to detect the serum IgG,intestinal,nasal,and lung sIgA antibody titers in chicks,and the challenge protection rate of chicks in the experimental group was evaluated.The results showed that the levels of IgG and sIgA antibodies in each immunized group were significantly higher than those in the control group(P<0.01).Compared with Twice immunized groups of pLA-F-S1/L.casei and three times immunized groups of pLA-F-S1/L.casei showed no significant increase in antibody titer,and the antibody duration was extended to the 28 th day after the third immunization.There were nosignificant differences in antibody levels among the pLA-F/L.casei group,the pLA-S1/L.casei group and the pLA-F-S1/L.casei group.The challenge test was carried out 7 days after the last immunization.The protection rates of chickens in the pLA-F-S1/L.casei group against NDV F48E8 and IBV M41 were 80% and 90%,respectively.The results of lymphocyte proliferation test showed that the level of cellular immunity was significantly increased after the recombinant recombinant bacteria,which was significantly higher than group of the pLA/L.casei(0.05 <p<0.01)and the group of PBS(0.01 <p <0.001).Therefore,combining the above results,pLA-F-S1/L.casei can induce mucosal immune response,cellular immunity and humoral immunity.It can effectively prevent infection of NDV and IBV.It is more economical than immunizing pLA-F/L.casei and pLA-S1/L.casei,respectively.It provides an important research basis for the development of oral vaccines for multivalent recombinant lactic acid bacteria against the disease.