Using Chicken GM-CSF To Enhance The NDV-F Antigen And Its Immune Effect As A Vaccine

Newcastle disease(ND)which caused by Newcastle disease virus(NDV)is a highly contagious,acute infection of poultry disease and it is one of the most harmful infectious diseases of Class A in poultry.F protein which is the major protective and virulence-associated antigens of NDV plays an important role in the production of the protective antibodies and immune responses in the body,and has become the first choice gene for developing ND vaccine.In recent years,GM-CSF as an effective immune-stimulating factor can enhance the immune response of DNA vaccines.However,the research on the effect of NDV vaccine related with GM-CSF is lagging behind.Therefore,chicken GM-CSF was detected in order to futher explore more effective NDV vaccine..In this study,the chicken GM-CSF and antigen F gene as the target genes were cloned to the baculovirus p LMI transfer vector which contains the CMV promoter,VSV-GED,WPRE and ITRs regulatory elements.Then the recombinant bacmid DNA were transfected Sf9 insect cells to obtain recombinant baculovirus through Bac-to-Bac system.The recombinant baculoviruses were immuned the chicken to effectively stimulate the body to induce antibodies strong humoral and cellular immunity to resist the NDV.GM-CSF was an adjuvant for enhancing immune effects of the NDV protective antigen protein.We compared and analyzed the effect of GM-CSF on the immune response.The recombinant baculovirus BV-LMI-F and BV-GM-CSF were Immunized the 14-day-old SPF chickens with different test groups,and set the commercial vaccines,vector and PBS control groups.Immune experiment results showed that immune protection ratio of F+GM-CSF group reached 100%,the immune protection ratio of F was 87.5%,the difference was signicant compared with the control group(p <0.05).This result domonstated that GM-CSF can be used as an immune stimulating factor to enhance the immune effect of the vaccine.The ELISA kits were used to detect the concentration of the Ig G,IL-2,IL-4 and IFN-? in the serum.At 56 days,the concentration of Ig G(1.70 VS 1.93)?IL-2(69.45 ng/m L VS 73.67 ng/m L)?IL-4(129.59 ng/m L VS 137.50 ng/m L)and IFN-?(65.30 ng/m L VS 69.15 ng/m L)were reached the highest level in the group F and F+GM-CSF,respectively.The difference was significant between the two groups(p <0.05).At the same time,we detected the level of neutralizing antibody and peripheral blood lymphocyte stimulus index to compare the immune effect of recombinant baculovirus vaccines.At 42 days,the level of neutralizing antibody was reached the highest value in F+GM-CSF group(1:1532)and it was sifnificantly different with F group(1:1317),p <0.05.In addition,flow cytometry was used to determine the concentration of CD4+ and CD8+ T cell in the blood.At 56 days,the level of CD4+ T cell were reached the peak in both F group(13.56%)and F+GM-CSF co-immunization group(15.66%),but it was significantly different between two groups,p <0.05.However,the level of CD8+ T cell levels was not significantly different between the groups.All above illustrated that recombinant baculovirus vaccine could stimulate the body to produce strong humoral immune and cellular immune response,and GM-CSF as an immune adjuvant could effectively improve the immunity effect.This study showed that chicken GM-CSF was a potential candidate of molecular immune adjuvant with a good adjuvant activity.At the same time,it also provided a new platform for developing a novel type of Newcastle disease vaccine.