Soluble Expression,purification, And Application Of Capsid Protein Of Very Virulent And Attenuated Strain Of Infectious Bursal Disease Virus

Infectious bursal disease?IBD?,caused by infectious bursal disease virus?IBDV?,is an acute and fatal infectious disease.IBDV mainly infects 3-6 weeks old chickens and targets the central immune organ-bursal of fabricius,causing heavy damage to B lymphocytes and triggering severe inflammation,which results in high mortality.The recover chickens often show grievous immunosuppression,leading to immune failure and increasing the susceptibility to other disease.The disease seriously threatens the healthy development of poultry farming.As the capsid protein of IBDV,VP2 is not only the main immunogen but also the important virulence protein.In addition,VP2 plays important roles in natural immunity.So VP2 is very critical in study on immune control and pathogenesis of IBDV.However,it was usually difficult to prepare the soluble viral capsid proteins with good bioactivity,which constrained the development of related studies.Gx,the reference strain of very virulent IBDV?vvIBDV?in China,is highly pathogenic to chickens;Gt,the attenuated strain derived from the Gx through continuous passage,is nonpathogenic to chickens and has been used as a vaccine strain.In this study,based on the prokaryotic expression system of Escherichia coli,the soluble expression of IBDV capsid protein VP2 was achieved under cold shock condition after a series of optimization.Furthermore,using affinity chromatography and gel filtration,VP2 proteins of very virulent and attenuated strain were efficiently purified.The obtained GxVP2 and GtVP2 proteins fused with His tag have good activity and can be self-assembled into IBDV virus-like particles?VLP?,which can react with IBDV standard positive sera specifically.After immunization to SPF chickens,both GxVP2 and GtVP2 stimulated good immune response,inducing higher titer of specific antibody and 90%protection against vv IBDV challenge.Compared with GtVP2 group,in GxVP2 group,higher titer of neutralizing antibodies were induced,no bursa atrophy in statistics was observed,?the average of BBIX is 0.81?,only mild lymphocyte necrosis was found in scattered folliculus.VP2 of vvIBDV has the potential value as a subunit vaccine.Host proteins interacting with VP2 play important roles in the pathopoiesia and immunization of IBDV,but their affinity has not been identified.In this study,using the macromolecule interaction instrument?Biacore T200,GE?which was characterized as"dynamic recording of molecular interaction process",the binding and dissociation process of GxVP2 or GtVP2 and Hsp90??viral cell-receptor?or CK1??innate immunity adaptor?were studied,their affinity was detected and compared.GxVP2 or GtVP2 could interact with host Hsp90?with similar magnitude,which was high affinity(10^-8 M).GxVP2 or Gt VP2 could interact with host CK1?with similar magnitude,which was moderate affinity(10^-4 M).In conclusion,the technical difficulty of the soluble expression of viral capsid protein was overcome,and the high purity VP2 proteins of very virulent and attenuated strain of IBDV were successfully obtained;both GxVP2 and GtVP2 have self-assembly activity,and vvIBDV VP2 has better immunological activity.The high affinity of VP2/Hsp90?and the moderate affinity of VP2/CK1?were determined,respectively.This study is of great significance for the study of IBDV immunoprotection and pathogenesis.