The Use Of Chicken IL-2 To Enhance The NDV-F Epitope And Its Immune Effect As A Vaccine

Newcastle disease(Newcastle diseases,ND),caused by NDV(Newcastle disease virus,NDV),a highly contagious,acute infection of poultry disease,is one of the most harmful infectious diseases of Class A in poultry.F protein is the major protective and virulence-associated antigens of NDV.It plays a so important role in produce protective antibodies and immune response in the body that it has become the first choice gene to develop ND vaccine.With the extensive application of monoclonal antibody technology,the F protein antigenic epitopes have been gradually recognized,Today the NDV antigenic variation is increasingly complex,it is crucial to choose the best appropriate and most protective immunity rate of antigenic epitopes to solve the problem of failure immune,which is cause by the antigenic differences in different virus strains.In recent years,As an effective immune-stimulating factor,IL-2 can be adjusted and enhanced DNA vaccine immune response,but the IL-2 of chicken(Interleukin-2,IL-2)research is lagging behind,in order to further explore the immunomodulatory effects to the NDV epitopes vaccine,the chicken restructuring IL-2(Interleukin-2,IL-2)is studied.In this study,The chicken immunostimulatory cytokines interleukin-2(Interleukin-2,IL-2),NDV major protective antigen F and the epitopes F_s,F_y gene were the target genes,the F_s and F_y gene were predicted by bioinformatics software,and cloned them to the baculovirus pLMI transfer vector with the CMV promoter and VSV-GED,WPRE,ITRs regulatory elements,then the recombinant Bacmid DNA were transfected Sf9 insect cells to obtain recombinant baculovirus by Bac-to-Bac system,the high titer recombinant baculoviruses were immune the chicken to effectively stimulate the body to produce antibodies to resist the NDV and strong response in cellular immune protection as the chicken vaccine,it was determined to screen the major protective antigen epitope gene of immunity for NDV F protein epitope further research foundation;At the same time,using the chicken interleukin-2(Interleukin-2,IL-2)as an immune-stimulating factor,the immune enhancement effects of different epitope vaccine were to compared and analyzed.it was foundation for enhance immune effects of the NDV protective antigen protein.The recombinant baculovirus BV-LMI-F,BV-LMI-F_s,BV-LMI-F_y and BV-LMI-IL-2 were successfully constructed,then the recombinant baculovirus were further infected the chicken embryo fibroblast cells.The SDS-PAGE and Western blotting results showed that can successfully detect the F protein,epitopes F_s,F_y protein and chicken IL-2 protein expression.Indirect immunofluorescence results showed:VSV-GED protein can be successfully expressed in Sf9 insect cells and show on the baculovirus surface to enhance the transduction efficiency of baculovirus.The recombinant baculovirus of BV-LMI-F,antigenic peptides BV-LMI-F_s,BV-LMI-F_y and BV-IL-2 were different combinations to immunize the14-day-old SPF chickens,and set the commercial of vaccines,vector and PBS control group immunized..Immune experiment results showed that immune protection ratio of recombinant bacillus vaccine group reached 100%,the immune protection ratio of F_s vaccine was 50%and F_y vaccine was 62.5%.compared to the control group,which immune protection ratio was only 12.5%(P<0.05).The immune protection ratio of F_s+IL-2 and F_y+IL-2 reached 62.5%and 75%respectively.IL-2 can be used as a immune stimulating factor,to enhance the immune effect of vaccine.The ELISA kits detected immune serum IgG,IL-2,IL-4,IFN-?,CD4~+molecular and CD8~+molecular levels,also detected immune serum level of neutralizing antibody and chicken peripheral blood lymphocyte stimulus index to comparison of the immune effect of recombinant baculovirus vaccine.the antigen of F,F_s and F_y recombinant baculovirus vaccine and immune group together with the chicken IL-2,F+IL-2,F_s+IL-2 and F_y+IL-2 recombinant baculovirus vaccine of serum antibody were significantly higher than that of PBS control group and empty vector group(P<0.05).At the 42 days,the F+IL-2 antibody reached the highest levels(1:1452)and it was significant difference to F antibody levels(1:1247)(P<0.05).At 42d,IL-2,IL-4 and CD4~+,CD8~+molecules concentration reached the 73.46 pg/mL,136.33 pg/mL and 79.83 ng/mL and 45.62ng/mL respectively,at 56d,the concentration of IFN-?reached up to 68.52 pg/mL,the IgG reached up to 1.846.It shows that recombinant baculovirus vaccine can stimulate the body to produce strong humoral immune and cell immune response.In conclusion,this study successfully constructed recombinant baculoviruses with F,F_s,F_y and IL-2 antigen gene,which can be highly effective expression in chicken embryo fibroblast.It has a higher transduction efficiency and longer expression time in the body than commercial vaccines Lasota.When the recombinant baculoviruses are co-immuned with IL-2,the body has a stronger immune effect.It is illustrated that the IL-2 can be used as an immune adjuvant.Recombinant baculovirus has better applicability than commercial vaccines Lasota.For the mutant strain,recombinant baculovirus can be quickly built according to the antigen gene of mutant strains to vaccine.This study will lay the foundation for the research of screening to determine more effective NDV protective antigen gene and it showed that chicken IL-2 has a good adjuvant activity,and is a potential application prospect of molecular immune adjuvant.At the same time,it also provides the new method for a new type of Newcastle disease vaccine.