Construction Of Recombinant Lactic Acid Bacteria Expressing Duck Tembusu Virus E Protein And Evaluation Of Immune Effects

Duck Tembusu virus disease is an emerging infectious disease of poultry caused by Duck Tembusu Virus(DTMUV),which is widely spread in Asian ducks and can cause viral encephalitis in ducklings and reduced egg production in laying ducks.Since Duck Tambusu virus infection was reported in China in 2010,the host has spread rapidly from ducks to chickens,geese,and birds.In recent years,there have been increased reports of DTMUV infection in chickens,which has increased the threat to the chicken industry,However,there is no specific vaccine against DTMUV infection in chickens.It is critical to developing a safe and effective DTMUV vaccine for chickens.Lactobacillus(LAB)is an important kind of intestinal probiotic,which can colonize and survive in the intestinal tract of animals,and can maintain the balance of intestinal flora,promoting digestion and absorption,and is also an excellent oral vaccine delivery carrier.Studies have shown that the fusion expression of dendritic cell-targeted peptide DCpep(DC-targeting peptide)and exogenous antigen in Lactobacillus plantarum can significantly improve the antigen presentation function of dendritic cells after oral immunization with animals,thereby improving the immune effect of the vaccine.The E protein is a capsule membrane protein on the surface of DTMUV,which has a variety of antigenic determinants that bind to host cell surface receptors and can stimulate the body to form antibodies.In this study,recombinant lactic acid bacteria NC8-p SIP409-pgs A’-E and NC8-p SIP409-pgs A’-DCpep expressing DTMUV E protein was established by anchored Lactobacillus plantarum expression vectors,and the immune protection effect against DTMUV infection was evaluated after oral immunization of chickens.The specific research content and results are as follows:1.Preparation of polyclonal antibodies against E proteinThe constructed prokaryotic expression strain BL21-p ET-28a-E successfully expressed E protein,SDS-PAGE results showed that E protein was expressed as inclusion body protein with a molecular weight of about 55 KDa.BALB/C mice were immunized to prepare polyclonal antibodies.Western-blot and indirect immunofluorescence detection showed that the prepared polyclonal antibodies could specifically recognize E protein and DTMUV.2.Construction of recombinant lactic acid bacteria expressing DTMUV E proteinThe amplified target fragments of E and E-DCpep genes were connected to the p SIP409-pgs A’vector to construct the recombinant plasmids p SIP409-pgs A’-E and p SIP409-pgs A’-E-DCpep,which were electrically transformed into Lactobacillus plantarum NC8 to obtain recombinant lactic acid bacteria NC8-p SIP409-pgs A’-E and NC8-p SIP409-pgs A’-E-DCpep,and cell membrane protein samples were obtained by the repeated freeze-thaw method,detection of E and E-DCpep protein expression by Western-blot,the results showed that E and E-DCpep proteins were successfully anchored on the membrane of Lactobacillus plantarum NC8.To detect the plasmid stability of recombinant lactic acid bacteria,it is continuously passed on for 50 generations,and dot plate counting every 10 generations,the plasmid stability was more than 90%.Finally,the growth performance of recombinant lactic acid bacteria was detected,and the results showed that the growth performance of NC8-p SIP409-pgs A’-E and NC8-p SIP409-pgs A’-E-DCpep was excellent.3.Evaluation of the immune protective effect of recombinant lactic acid bacteriaThe results of animal experiments showed that:After immune recombinant lactic acid bacteria,the percentage of CD3~+CD4~+and CD3~+CD8~+T lymphocytes in peripheral blood and spleen of pgs A’-E group and pgs A’-E-DCpep group was significantly higher than that of the control group,and the percentage of CD3~+CD4~+and CD3~+CD8~+T lymphocytes in peripheral blood and spleen of pgs A’-E-DCpep group was significantly higher than that of pgs A’-E group.Using E protein to stimulate splenocytes,it was found that the pgs A’-E group and the pgs A’-E-DCpep group significantly promoted the proliferation of splenocytes.ELISA results showed that the content of specific Ig G and mucosal antibody s Ig A in pgs A’-E group and pgs A’-E-DCpep group were significantly higher than those of the control group(P<0.001,P<0.001),and the contents of cytokines IL-2 and IFN-γwere significantly higher than those of the control group(P<0.001,P<0.001),indicating that after oral immunization with the two strains of recombinant lactic acid bacteria in the chickens,they could improve the body’s cellular immunity,humoral immunity,and mucosal immune response,and promote the expression of cytokines IL-2 and IFN-γ.In the challenge protection experiment,the net gain of body weight in the pgs A’-E group and pgs A’-E-DCpep group was higher than that in the pgs A’group and PBS group.The infection rate and organ tissue toxicity in the pgs A’-E group and the pgs A’-E-DCpep group were significantly lower than those in the PBS group,and the organ index was not significantly different compared with the Control group.These results indicated that NC8-p SIP409-pgs A’-E and NC8-p SIP409-pgs A’-E-DCpep have certain anti-DTMUV infection abilities.In summary,this study constructed an anchored Lactobacillus plantarum expressing DTMUV E protein,which can effectively enhance the cellular immunity,humoral immunity,and mucosal immune response of chickens,promote the expression of cytokines IL-2 and IFN-γ,improve the ability of immune chickens to resist DTMUV infection,and provide a reference for continuing to explore the new DTMUV vaccine in chickens in the later stage.