The Sequencing Of Infectious Bursal Disease Virus GXBH Genome And Identifiction Of The Cellular Proteins Binding To Structural Protein VP3

Infectious bursal disease virus(IBDV),primarily affects chicks’ immune systems,and is the etiological agent of infectious bursal disease(IBD)which causes a severe and highly contagious disease in chickens,leading to severe damage to the chicken industry.In the present study,we sequenced the genome of IBDV strain GXBH and assessed its pathogenicity in chickens.We also developed a monoclonal antibody against VP3 protein and used it as a coupling antibody in immunoprecipitation to discover the host proteins that interact with the VP3 protein.The results are as follows:1.Genomic sequencing and pathogenicity assessment of IBDV strain GXBH:We isolated an IBDV strain(designed GXBH)from 20-day-old chicks with clinicopathological signs associated with infectious bursal disease in Guangxi region.This IBDV is a recombinant strain with fragments A and B being from a recombinant intermediately-attenuated vaccine strain and a classical virulent strain,respectively.Necrosis,lymphocyte depletion,and lymphoid follicle atrophy were seen in the bursa of Fabricius of chickens when inoculated with the IBDV strain GXHB.2.Preparation of monoclonal antibody against VP3 protein:The VP3 gene was amplified and inserted into prokaryotic expression vector pET-28a,transformed into E.coli BL21,and the recombinant protein was obtained by IPTG induction.The purified recombinant protein was immunized to mice,and the monoclonal antibody hybridoma cell line of VP3 protein was obtained by cell fusion,positive screening and subcloning.The monoclonal antibody was identified as IgM for the heavy chain and Kappa for the light chain by subclass identification.3.Identification of VP3 protein-interacting host proteins:Using the monoclonal antibody prepared above,a total of 155 potential host proteins capable of interacting with VP3 protein were screened by a combination of immunoprecipitation(Co-IP)and liquid-phase mass spectrometry(LC-MS)techniques;KEGG analysis showed that the host-interacting proteins are mainly involved in biochemical metabolic pathways and signal transduction pathways,including constitutive ribosomes,spliceosomes,RNA transport,gap junctions,tight junctions,insulin signaling pathway,and mTOR signaling pathway.DDX21,DDX6,PABPC4,DDX18,and MOV10 were selected as candidate host-interacting proteins,and these candidate proteins were further identified as interacting with VP3 proteins after Co-IP validation.In conclusion,in this study,a recombinant strain of chicken infectious bursal disease virus was isolated and identified from Guangxi region,and a monoclonal antibody to VP3 protein was obtained.Using this monoclonal antibody to identify the host protein that interacts with VP3 protein will lay the foundation for further research on the pathogenic mechanism of IBDV and the development of effective prevention and control technologies.