The Immunogenicity Comparison Between VLP Of Very Virulent And Attenuated Strain Of IBDV And Construction Of Phage Display Library Of Antibodies Against IBDV

Infectious bursal disease(IBD)is a disease caused by infectious bursal disease virus(IBDV),which mainly attacks bursa of chicken,an important central immune organ,and can cause immunosuppression.In clinic,IBDV infection leads to severe secondary infection,causes the death of chickens and endangers the development of poultry farming.Vaccine is an effective method to prevent and control IBD.Traditional vaccines have some unavoidable shortcomings.Widespread application of attenuated virus vaccines can lead to mutations of virus,while inactivated vaccines of virulent virus have biological safety risks such as incomplete inactivation.Subunit vaccines such as virus-like particles(VLPs)can overcome these shortcomings and induce the body to produce a good immune response.At present,many poultry farms also use egg yolk antibodies to treat IBD-infected chickens to reduced economic losses.Although the preparation of yolk antibodies is simple,it has the disadvantages such as low purity and low antibody titer.Therefore,using genetic engineering methods to express highly purified therapeutic antibodies is the trend of producing therapeutic antibodies.VP2 is the only capsid protein of IBDV,which can stimulate the body to produce neutralizing antibodies and determine the antigenicity of IBDV.It is an important protein for the preparation of VLP vaccines,but there are differences in the immune response induced by VP2 from IBDV very virulent and attenuated virus.In order to screen more effective IBDV VLP vaccine candidates,on the basis of the preparation of a very virulent IBDV strain called Gx strain,the VP2 gene sequence of the attenuated IBDV strain called Gt strain was amplified and cloned into a yeast expression vector,pAO815.After the pAO815-NHisGtVP2 plasmid was electroporated into the yeast strain SMD1168,the VP2 protein was successfully expressed after inducing for 5 days.Purified attenuated VLP of IBDV was prepared by hydrophobic chromatography.At the same time,a more efficient VLP purification method was established.Agar diffusion test results show that Gt VLP has good antigenicity.The SPF chickens were immunized with Gx and Gt VLPs and their immune protective effects against IBDV were compared.The neutralizing antibody titers in this two immunization groups have been at a high level within 5 weeks after immunization,indicating that both of them could induce a sustained immune response.The virulent VLP immunization group could produce 100% attack protection,higher than the attenuated VLP immunization group which produce 80% of the attack protection rate.The bursa of chicken in attenuated VLP immunization group atrophied significantly after the attack.These results show that the Gx VLP has a better protective effect on chicken than the Gt VLP.Phage display technology is an effective technique for screening of antibodies.In order to screen efficient therapeutic antibodies of IBD,we isolated peripheral blood lymphocytes from whole blood of chickens immunized with Gx VLPs and amplified the light chain variable region(VL)and variable region of heavy chain(VH)sequences.The pCANTAB5E-scFv plasmid was constructed by cloning VH and VL into phagemid,and then the anti-IBDV phage antibody library was established after transformation.Through three rounds of panning enrichment and ELISA identification,we got five positive antibody sequences.Five activities neutralizing activity in vivo and in vitro will be verified in the next step.In this study,we compared the immunogenicity of very virulent and attenuated virus-like particles of IBDV,our findings provided an important reference for the clinical research and application of subunit vaccines.Additionally,we established a anti-IBDV phage antibody library and obtained five antibody sequences for efficient IBDV,which laid the foundation for the screening and expression of efficient antibodies.