Immune Enhancement Of Recombinant Chicken Interleukin-7 For Chicken Infectious Bursal Virus Inactivated Vaccine

Infectious bursal disease(IBD)is an acute contagious disease caused by IBD Virus(IBDV).So far it has not been effectively controlled and brought great economic loss to the poultry industry.At present,the preventive vaccination is main strategy to be adopted to prevent this disease.Currently used vaccines include live attenuated vaccine and inactivated vaccine.The former has good immunization effect,but hides the potential danger of virulence reversal of vaccinated virus and occurrence of subclinical symptoms;although the latter has low toxic-side effects,its immunogenicity is relative low.Therefore,to develop adjuvant for IBDV inactivated vaccine to enhance its immunogenicity is crucial for effective prevention of IBDV.In recent years,the cytokine-based biological adjuvants are widely investigated since the cytokine adjuvants not only have natural immunoregulatory functions,but also have the characteristics of low toxic-side effects and high biological safety,which can significantly enhance the immune effects for many vaccines.Interleukin-7 is an important multifunctional cytokine,it not only promotes the generation and development of B cells and T cells,but also plays a role in coordinating other cytokines to improve animal immunity.We have previously demonstrated that interleukin-7(IL-7)could significantly improve the immunogenicity of DNA vaccine of canine parvovirus,meanwhile,the recombinant chicken IL-7(chIL-7)was identified to have ability to stimulate anti-IBDV activity of chickens,and improve the immunogenicity of IBDV VP2 DNA vaccine.However,whether recombinant ch IL-7 is able to enhance the immunogenicity of IBDV inactivated vaccine is not clear.Therefore,in this study,the immune enhancement of chIL-7 on IBDV inactivated vaccine was analyzed by preparing recombinant chIL-7.First,the chIL-7 gene was amplified from the plasmid containing the chIL-7 cDNA gene by PCR and inserted into the prokaryotic expression vector pET-20b(+)to generate chIL-7 prokaryotic expression vector pET20b-chIL-7/H,in which the chIL-7 gene was fused with His-tag.The expression vector was transformed into E.coli BL21 to produce engineered bacteria and the recombinant chIL-7 was expressed in the E.coli under the IPTG induction.The expressed chIL-7 was purified by Ni-NTA agarose beads,and identified by Wsetern-blot.The biological activity of chIL-7 was analyzed by 2E8 cell proliferation assay.Then,the chickens were co-immunized with IBDV inactivated vaccine and the recombinant chIL-7 at different doses,and then boosted once at one week after the first vaccination.Blood samples were collected before and after the first immunization respectively and serum was separated.The antibody titer and neutralization antibody titer were detected by ELISA.The stimulation index of spleen lymphocytes in chicken at 30 d after immunization was detected by MTT,and the levels of interferon-? and IL-4 were detected by ELISA.The immunized chickens then were challenged with a virulent IBDV strain.At 8 d after the challenge,the mortality and survival rates of the immunized chickens were calculated in order to evaluate the chIL-7 capacity for enhancement on immunogenicity of the IBDV inactivated vaccine.The results showed that the constructed expression vector could mediate the expression of chIL-7 gene in E.coli BL21,and the expressed chIL-7 had the biological activity.The immunization results showed that the antibody titer and neutralization antibody titer in the sera of chIL-7/IBDV vaccine co-immunized group were significantly higher than those in the IBDV vaccine immunized group,and the lymphocyte stimulation index and interferon-? level in the co-immunized group were also significantly higher than those in the IBDV vaccine immunized group.The challenge results showed that the survival rate(91%-97%)of the co-immunized group was significantly higher than that of the IBDV vaccine immunized group(78%).In addition,the index of bursa of Fabricius,the scores of pathological lesion and the IBDV titer in the infected tissues in co-immunized group were lower than those in the IBDV vaccine immunized group.It can be seen that recombinant chIL-7 can significantly enhance the immunogenicity of IBDV inactivated vaccine and improve its protection rate of the immunized chickens.This study laid the foundation for the further application of recombinant chIL-7 as a biological adjuvant in IBD disease prevention.