Construction Of A Multi-epitope Targeted Vaccine Against Foot-and-mouth Disease And Effect Of Mucosal Immunity

Foot-and-mouth disease(FMD)is an acute,febrile and highly contagious infectious disease caused by foot-and-mouth disease virus(FMDV),which mainly affects livestock such as pigs,cattle,sheep and other wild cloven-hoofed animals.Its clinical features are vesicular rash in oral mucosa,hoof and breast skin.The World Organization for Animal Health(OIE)lists FMD as an animal class A severe infectious disease,because of FMD serious harm to the healthy development of animal husbandry and the foreign trade of related products.This situation has far-reaching influence on the politics and economy of the country.Mucous membrane,as a gateway to the outside world,plays an important role in the invasion of FMDV.Mucosal immune system,as the first barrier against the invasion of pathogenic microorganisms,plays an vital role in the infection and invasion of FMD.Microfold cell(M cell)is the first cell in mucosal immune response,which is the initial effector site of mucosal associated lymphoid tissue.The uptake of antigen is the most critical step in inducing immune response,so M cell plays an important role in pathogenic microorganism invasion from respiratory tract and mucosal immune presentation antigen.In this study,two recombinant Lactococcus lactis strains named NZ9000/pNZ8148-TB1 and NZ9000/pNZ8148-TB1-Co1 were successfully constructed in order to study the immune effect of recombinant Lactococcus lactis and the feasibility of targeting M cells by using the target protein of Lactococcus lactis expression system(NICE system).It provides a new way for the development of a new vaccine against FMD and the prevention and treatment of FMD.The main contents of this study are as follows:1.Synthesis and Bioinformatics Analysis of TB1 and TB1-Co1:The coding genes of multi-epitope TB1 and TB1-Co1 were optimized and synthesized according to the preference of Lactococcus lactis codon,and then the genes were added to the cloned vector pUC57.Multi-epitope TB1 and TB1-Co1obtained through bioinformatics have the potential to be good antigens,laying a theoretical foundation for further research.2.Expression and identification of the target protein in Lactococcus lactis:The target gene fragment TB1 and TB1-Co1 were connected to the expression vector pNZ8148,and the recombinant plasmid pNZ8148-TB1 and pNZ8148-TB1-Co1 were transformed into Lactococcus lactis NZ9000 by electroporation.The recombinant strains NZ9000/pNZ8148-TB1 and NZ9000/pNZ8148-TB1-Co1 were obtained by plasmid extraction,double enzyme digestion and sequencing analysis.The target proteins were induced and expressed by inducer nisin,and the expression conditions were optimized.Western blot analysis showed that the target protein was expressed successfully in Lactococcus lactis NZ9000.3.Analysis of mucosal immune effect of recombinant Lactococcus lactis(NZ9000/pNZ8148-TB1 and NZ9000/pNZ8148-TB1-Co1):Immunogenicity analysis was performed on mice immunized with recombinant Lactococcus lactis by gavage.Meanwhile,PBS was used as blank control,NZ9000/pNZ8148 as blank vector control,and inactivated vaccine as positive control.The results showed that the level of sIgA in intestinal mucus of experimental group was significantly higher than that of negative control group and empty vector control group,but the titer of specific IgG antibody in serum was lower than that of control group.The ratio of CD4~+cells to CD8~+cells in the lymphocytes of the experimental group was higher than that of the negative control group,and the proliferation of the spleen lymphocytes of the mice was obvious after antigen stimulation.The results of cytokine assay showed that the levels of IL-2,IL-4,IL-5,IL-10 and IFN-?in the experimental group were higher than those in the negative control group.These results suggest that recombinant Lactococcus lactis can induce FMDV specific humoral and cellular immune responses in mice as an oral live vector vaccine.4.Target experiments:The mice were inoculated with recombinant Lactococcus lactis NZ9000/pNZ8148-TB1-Co1 and NZ9000/pNZ8148-TB1.The small intestine tissue of the mice was made into frozen sections and observed by Confocal Laser Scanning Microscope after fluorescence staining.The results show that the binding of red fluorescence and green fluorescence in the former is more than that in the latter.This suggests that Co1 has a targeted effect on M cells and can enhance the immune response of the body.