| Classical swine fever(CSF)is a devastating infectious disease caused by classical swine fever virus(CSFV),which poses huge economic losses to the pig industry in China and even the world.At present,CSF is endemic in China,and vaccine immunization remains the main measure to prevent,control and eliminate CSF.The well-known lapinized Chinese strain of CSFV(C-strain)is safe and efficacious in the prevention and control of CSF.However,C-strain cannot distinguish wild-type virus infection from vaccine-immunized animals,which is not conducive to the eradication of CSF.In addition,conventional CSF vaccines such as C-strain and subunit vaccines are often immunized via intramuscular injection,which is complicated to operate,easy to contaminate,produce stress reaction,and cannot meet the immune needs of wild boars.The oral vaccine is convenient to immunize by the oral route,which can not only induce systemic immune response,but also induce local mucosal immunity.In the development of oral vaccine,lactic acid bacteria(LAB)are ideal mucosal immune delivery vehicle for vaccine antigen presentation.As an important symbiotic probiotic in the gastrointestinal tract of the body,LAB can resist the erosion of the acidic environment and bile salts of the gastrointestinal tract,and deliver the antigen to intestinal immunity induction site,while maintaining the dynamic balance of the intestinal flora of the body and promoting the absorption of animal nutrients.In this study,two strains of recombinant Lactococcus lactis(L.lactis)were constructed using food-grade induced expression system NICE,which fused CSFV E2 protein with RCK or OmpH protein respectively,named rL.lactis-E2-RCK and rL.lactis-E2-OmpH.The two strains of recombinant L.lactis successfully expressed fusion proteins of 42 kDa and 39 kDa,respectively.In addition,the immune efficacy of recombinant L.lactis was evaluated in rabbits.The results showed the rabbits immunized with the rL.lactis-E2-RCK and rL.lactis-E2-OmpH did not developed CSFV-specific IgG,sIgA and neutralizing antibodies.The r L.lactis-immunized rabbits were not protected from fever induced by C-strain.Then,we selected the carrier of Lactobacillus plantarum(L.plantarum)with stronger colonization ability and longer colonization time to enhance the uptake of intestinal mucosal antigen and induce an effective immune response.We constructed a recombinant L.plantarum NC8-pSIP409-E2 expressing the CSFV E2 protein.The mice were orally immunized with NC8-pSIP409-E2,and serum IgG and fecal sIgA antibodies were detected by ELISA.The results showed the specific IgG antibody was negative,but the level of the sIgA antibody in feces of the experimental group was higher than that of the control group.T lymphocyte subtype analysis and lymphocyte proliferation test were performed.Compared with the control group,the proportion of CD4~+and CD8~+T cells and lymphocyte stimulation index increased significantly.Finally,in order to find a suitable LAB carrier to optimize the expression and delivery of E2 antigen protein,three strains of wild boar-derived LAB were isolated in this study,and their initial research and safety evaluation for carrier development were performed.It is hopeful to transform them into oral vaccine carriers to induce high-level immune responses.In summary,this study successfully constructed recombinant L.lactis and recombinant L.plantarum expressing the CSFV E2 proteins,and further immunogenicity evaluation confirmed that the rabbits immunized with the recombinant L.lactis were not protected from the fever induced by C-strain and the recombinant L.plantarum induced cellular immunity in mice.In addition,the isolated three strains of wild boar-derived LAB have the potential to be used as oral vaccine delivery carriers for the development of oral CSF vaccines. |
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