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Research of Binding Epitopes on VP2 Protein of IBDV to B Cell Receptors

Author: LiZuo
Tutor: WangAiPing
School: Zhengzhou University
Course: Cell Biology
Keywords: Infectious Bursal Disease Virus VP2 protein binding epitope T7 phage display overlapping polypeptide scanning
CLC: S852.65
Type: Master's thesis
Year: 2008
Downloads: 89
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Abstract


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 responsible for severe immunosuppressive disease that causes significant losses to the poultry industry.VP2 protein is the main capsid protein of IBDV,which composes the surface of virus particles,and contains the antigen epitopes.Researches show that mutations of animo acid 253,279 and 284 not only lead to the difference of virulence among virus,but also cause the adaptation to chick embryo fibroblases(CEFs) and Vero cells of non-virulent IBDVs but not virulent IBDVs.It is presumably that the affinity between VP2 protein and cell receptor has been changed by mutation of the three animo acids.To reveal the interaction between VP2 protein and host cells of IBDV,we separated VP2 protein into 6 overlapping fragments by PCR amplification.Each fragment was displayed on T7 phage surface,as well as complete VP2 protein. Recombinated phages were detected,and then labelled by FITC after being purified by PEG precipitation.Prepared T7 phages were interacted with B cells of 3～6-week-old chicken brusal and Vero cells.Results showed that T7phages which displayed VP2 protein,B,B1,B2 and B3 fragments can bind to both B cells and Vero cells,but the affinity to B cells is much stronger,while both have high affinity to VP2 protein and B2 fragment.The conclusion is as follows:the hypervariable region of VP2 protein(B fragment) can bind to receptors both on B cells and Vero cells;B2 fragment(contains amino acid 253,279,284) is important to this interaction.The binding epitopes of VP2 protein may separate on its hypervariable region,and aggregate through protein folding,but no denying the probability that binding epitopes are only on B2 fragment,the binding properties of B1 and B3 fragments owning to the overlapping part of B2 fragment.In order to verify the binding properties to cell receptors,vp2 and its fragments were cloned in eucaryotic expression vector pEGFP-C1.After stable transfection, recombinated proteins were purified by Ni-NTA affinity column,and then interacted with B cells and Vero cells.No green fluorescence were observed or detected in both experiments.The reason may be as follows:GFP was inactivated while purification, or interested proteins were too few to bind to the cell receptors.Polypeptide scanning can be used as a simple and efficient way to study epitopes or interactions between ligands and receptors.Based on the bioinformatic analysis of space structure of B2 fragment,6 overlapping polypeptides were designed and synthesized by solid phase peptide synthesis,and were uesd to block the interaction between T7 phage that displayed VP2 protein and receptors on B cell and Vero cells. Results showed interactions can obviously be inhibited by polypeptides 5t and 6t in both cells with the consentration of 125μg/mL.Both polypeptides contain critical amino acid recidue,284T,which may play a major role in binding to receptors on both cells.284T is also contained in polypeptide 4n,which has much longer lateral sequence and lower affinity to cell receptors.Results also showed 1h can weekly bind to B cells,but did not show in Vero cells.In summary,it is indicated initally in this research the possible regions on VP2 protein of IBDV that may include in binding to receptors on B cells and Vero cells. These findings may provide a theoretical basis for further studies in interaction between virus and cell receptors,and production of vaccines and medicines for IBD.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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