Enhancing Immune Effect Of The Recombinant Fusion Peptide (T?1-BP5) To DTUMUVA Inactivated Vaccine

Duck Tembusu Virus(DTMUV)is a new type of flavivirus.After infection the main feature is a serious decline in egg production.It's wide range,rapid spread.The duck present as high fever in the early stage,and a certain proportion of neurological symptoms in the late onset,mainly show as rolling,paralysis,standing instability and ataxia,and some even died quickly in few days.At present,the prevention of the disease mainly through immunize the Duck Tembusu Virus inactivated vaccine,but the inactivated vaccine immune effect still needs to be improved,the prevention and control of the disease still need to be strengthened.In this study,two proteins capable of specifically binding to the E protein of DTMUV HN1 strain were screened by phage display technology using DTMUV HN1 strain E protein as target,and detected its inhibitory effect on DTMUV.At the same time,genetic engineering was used to synthesize recombinant fusion peptide T?1-BP5(rT?1-BP5)and detect its expression.Finally,ducklings were immunized with rT?1-BP5 combined with DTMUVA inactivated vaccine,analyzed its immunize effect,aimed to lay a foundation for the prevention and control of duck disease.The main contents are divided into the following three aspects:1.Screening and Identification of Specific Binding Peptides to Duck Tembusu Virus E Protein by Phage Display Peptide LibraryE protein was used to screen its binding peptides from phage display peptide library,two positive phage clones were found and the sequencing results showed which encoded the peptides of HWSTRQGSTRWN(P3-12),and THRSWQGNSWYM(P8-12).In addition,the peptides P3-12 and P8-12 were synthesized and tested in duck embryo fibroblast.The results indicated that E protein inhibitory peptide had no significant effect on the proliferation of duck embryo fibroblast,and inhibitory peptides of different concentrations were significantly down regulated by DTMUV and can significantly reduce the virus titer of DTMUV in duck embryo fibroblast.In conclusion,this study provide some reference data for further study on the mechanism of Duck Tembusu Virus inhibitors.2.Fusion Expression of Thymopentin ?1(T?1)and Bursopentin(BP5)Recombinant Fusion Peptide and Its Adjuvant EffectGene of T?1-BP5 was synthesized and used MTT to detect its expression.To evaluate the immune adjuvant properties of rT?1-BP5,four experimental groups were set up with PBS control group,mice were immunized with rT?1-BP5 combined with inactivated H9N2 AIV vaccine.The titers of HI antibody,antibodies(IgG1 and Ig G2a)sub types,and levels of Th1-type cytokines(IFN-?)and Th2-type cytokines(IL-4)were assessed subsequently;meanwhile,its immune protective effects were estimated by virus challenge experiment.The results showed that The titer of HI antibody and the levels of IgG1 and IgG2 a inrT?1-BP5 group were significantly higher than those in other groups,and the levels of IL-4 and IFN-? in serum were significantly higher than those in other groups.The virus titer in the lungs of the mice was significantly reduced and significantly different from the other groups,and in 7d,almost no virus titers were detected in the lungs.It showed that rT?1-BP5 could enhance HI and IgG sub-type antibodies titers,promote the secretion of Th1-and Th2-type cytokines,which suggested that r T?1-BP5 could enhance the humoral and cellular immune responses.Detection results of virus of challenged mice in lungs showed that rT?1-BP5 could contribute to the removal of lungs virus in mice.This study lays a foundation for the further work to research and develop a new vaccine adjuvant.3.Enhancing Immune Effect of The Recombinant Fusion Peptide(T?1-BP5)to DTMUVA Inactivated VaccineIn this study,ducklings were immunized with rT?1-BP5 combined with DTMUVA inactivated vaccine to detect the level of IgG antibody,serum cytokine level.Virus challenge experimentwere and HE staining texted to evaluate the immune protection of ducklings.The results showed that the levels of serum IgG antibody,cytokines(IL-4,IFN-?)in the vaccine group were significantly higher than those in the control group and the vaccine group.Virus challenge experiment results showed that rT?1-BP5 could enhance the immune response of ducklings and protect the duck liver from damage.This study laid the foundation for the research of immunological adjuvants for DTMUVA inactived vaccine.