Construction Of A Chicken B Cell CDNA Library Displayed On Phage T7 And Identification Of B Cell Receptor For Infectious Bursal Disease Virus (IBDV)

Infectious bursal disease(IBD)is a viral disease of young chickens characterized by necrosis and depletion of lymphoid tissues,especially the bursa of Fabricius.The causative agent of this disease,IBD virus(IBDV)is a member of the genus Avibirnavirus of the family Birnaviridae which consists of double-stranded RNA genome and responsible for severe immunosuppressive disease that causes significant losses to the poultry industry.As with all viruses,IBDV needs to penetrate target cells to cause infection by a communication of the receptor binding.The attachment of the virus to a specific receptor on the surface of susceptible host cells is the first step in virus infection and has attracted attention as targets for infection prevention.It is of importance to study the virus infection at the molecular level of virus binding for understanding the virus-host cell interaction and pathogenesis of IBD.However,the investigation on the complex biochemical processes involving virus binding to host cell surface molecules is hindered by the lack of knowledge on the identities of the virus receptor of IBDV.To identify the receptor which may determine the susceptible B lymphocyte binding and viral infection,a chicken B cell cDNA expression library displayed on phage T7 was constructed via phage display technique.Chicken Igλlight chain was identified after the library secreening and DNA sequencing.Virus binding and inhibition assay confirmed that the IBDV-nonpermissive COS7 cells bound viruses specifically once the candidate receptor was expressed on the cell membrane of COS7.Data suggest that chicken Igλlight chain is a binding receptor for IBDV on B cells and facilitate virus binding to B cells.1 Construction and quality identification of chicken B cell cDNA expression library displayed on Phage T7To construct the chicken B cell cDNA expression library displayed on phage T7 and to identify its quality,mRNA was prepared from bursa of Fabricius in a SPF chicken by oligo(dT)-cellulose affinity chromatography and reverse transcripted into cDNA.A chicken B cell cDNA expression library was constructed after cloning cDNA into T7 EcoRI/Hindâ…¢vector arms and in Vitro packaging.Titer and recombinant rate of the prime library were determined.The size of the inserts was identified by PCR after a round of library amplification.The titer of the prime constructed library and amplified library was 5×10⁷ pfu/mL and 1.88×10¹⁰pfu/mL.The inserts size largely ranged from 0.75 to 2kb with the average length of 1.44kb.The chicken B cell cDNA expression library displayed on phage T7 has high quality.2 Screening the T7 expression library for phage clones with high affinity for IBDVPurified IBDV was used as the bait in a biopanning procedure.Immobilized IBDV was probed to bind T7 phages that were expressed proteins from a chicken B cell cDNA library.The phage recovery percentage was assessed by output/input phages as a measure of enrichment and determined rounds of biopanning.After four rounds of biopanning,the bound phages were isolated and their affinity and specificity for IBDV was detected by plaque lift and phage ELISA.80 candidate clones were sequenced and analyzed.Most sequences of the clones containing that may be involved in IBDV binding to B cells were chicken Ig lambda light chain sequences from open reading frame of the protein.The sequences show high identities to chicken Ig lambda light chain in GeneBank.3 Expression of recombinant chicken Igλlight chain in virus nonpermissive COS7 cellsRecombinant DNA ecoding chimeric protein incorporating transmembrane region of bovine IgG Fc receptorγRâ…¡(bFcRγRâ…¡)and chicken Igλlight chain was generated by fusion PCR.pEGFP-C1-SP,the vector with signal sequence was then created by fusing signal peptide of chicken Igλto GFP N terminus.The chimeric full length sequence was respectively cloned into modified pEGFP-C1-SP and pcDNA3 plasmid using double restriction sites.Mammalian expression vector pEGFP-C1-SP-λR2T and pcDNA-λR2T ware successfuLly constructed.The chimeric transmembrane protein was located principally on the plasma membrane in COS7 cells transfected with vector pEGFP-C1-SP-λR2T under fluorescent microscope or with vector pcDNA-λR2T by flow cytometric analysis.The secretable expression of chicken Igλwas observed under fluorescent microscope and in Western blotting by inserting the chicken Igλgene into eukaryotic expression plasmid pEGFP-C1-SP and pcDNA3 and then transfecting the recombinant vector into COS7 cells via lipofectamin.4 Biological function of chicken Igλlight chain as a cellular receptor for IBDV recombinant vector into COS7 cells via lipofectamin.4 Biological function of chicken Igλlight chain as a cellular receptor for IBDVTo examine whether the recombinant chicken Igλcould interact with IBDV,VOPBA was performed.The purified IBDV bound to recombinant chicken Igλwas detected with MAb against IBDV nucleocapsid protein,while no band was observed in negative controlTo determine function of chicken Igλlight chain as a IBDV receptor that can facilitate IBDV binding to nonpermissive cells,COS7 were transfected with plasmid constructs coding for chimeric transmembrane protein and exposed to virus on ice to allow virus binding.COS7 cells transfected with chimeric construct showed high levels of virus binding in comparison to nontransfected COS7 cells.The binding of IBDV occurred in a dosed-dependent manner.COS7 cells without expression of chicken Ig lambda light chain and lymphocytes from the bursa of Fabricius of 4-week-old chicken were exposed to virus,based either on immunofluorescence microscopy or flow cytometry using a anti-IBDV antibody.BF lymphocytes showed high levels of virus binding,in contrast,very little binding was observed with COS7 cell control.For competition of IBDV binding the viruses were incubated with recombinant chicken Igλlight chain or antiserum specific for IBDV before incubating with cells. Inhibition of virus binding was obtained by incubation of cells with mAb against chicken Igλlight chain before being tested for the ability to bind viruses.Detection of bound viruses was performed by flow cytometry.The inhibition effects on virus binding were much more obvious than that in control.These results demonstrated that chicken Igλlight chain may act as a cellular receptor facilitating IBDV binding to chicken B lymphocytes. The findings in this study provide a basis for further exploration of molecular mechanism by which IBDV infects chicken B lymphocytes.