Epitope Mapping For Specific Proteins Of Retroviruses That Induce Diseases In Poultry

The main species of avian retrovirus cause neoplasms in poultry is the Avian Leukosis virus(ALV)and Reticuloendothelial virus.The ALV-p27 protein,which is encoded by the gag gene,is the capsid protein and acts as a group-specific antigen.The ALV-p27 gene is highly conserved and exhibits 96%sequence identity among the exogenous subgroups(A,B,C,D and J).In addition,the p27 protein content accounts for more than 30%of the viral protein,and it has many viral antigen epitopes that are easy to detect.The gp90 protein of REV is associated with virus neutralization,which is known to be the major candidate antigen for vaccines and disease serological diagnosis.The different REV strains show only minor variations in the genetic sequences,and they are antigenically similar,which suggested that a subunit vaccine expressing the gp90 gene of a single REV isolate may provide protective immunity against numerous REV-associated diseases.Detailed analysis of epitopes is important for the understanding of immunological events,and the development of epitope based marker vaccines and diagnostic tools for various diseases.This thesis,based on research presents results from using overlapping peptides method and microarray method for identifying the recognition site of monoclonal antibodies on their antigens.Firstly we describe the identification of a novel linear B-cell epitope of ALV.Epitope mapping for ALV-p27 was performed using mouse monoclonal antibody(5D3).ALV-p27 was translated into 5 overlapping fragments amplified by PCR from ALV-J strain JS-nt,cloned into the pGEM-T easy vector,Then amplified from this recombinant vector.After that,these fragments were subcloned into the pGEX-6P-1 vector and a collection of expression clones that corresponded to the desired fragments were constructed and the recombinant proteins were subjected to western blotting analysis to determine the immunodominant region.For the precise mapping of this epitope,series of 11 overlapping peptides,which covered 173-240 amino acids of ALV-p27 protein,were synthesized.Indirect ELISA showed that the CFRQKSQPDI,which corresponds to 193-202 aa of ALV-p27 was the minimal requirement for reactivity of 5D3.The motif was the minimal requirement for reactivity as demonstrated by analysis of reactivity of 5D3 with several truncated peptides that derived from the motif.Comparison of 5D3 defined epitope sequence(193CFRQKSQPDI202)with the sequences of other ALV strains indicated the epitope is highly conservative among ALV strains.Moreover,we illustrated the identification of linear B-cell epitopes recognized by two monoclonal antibodies(mAbs)6C12 and 09980 against the REV-gp90 using peptide microarray.REV-gp90 was translated into linear 15-mer overlapping peptides with a peptide-peptide overlap of 14 amino acids.The gp90 peptide microarray copies were incubated with the mAbs at a concentration of 1 ?g/ml in incubation buffer followed by staining with the secondary goat anti-mouse IgG(H+L)DyLight680 antibody and read-out with a LI-COR Odyssey Imaging System.Quantification of spot intensities and peptide annotation were done with PepSlide(?)Analyzer.Mouse monoclonal antibody(6C12)showed a strong and clear IgG response against an epitope-like spot pattern with the consensus motif YHPLA which is corresponds to 216 aa to 220 aa of gp90 protein sequence.On the other hand mouse monoclonal antibody(09980)showed a very strong and clear IgG response against an epitope-like spot pattern with the consensus motif DPQTSDILEA which is corresponds to 230 aa to 239 aa of gp90 protein sequence.Further identification of these B-cell epitopes was conducted using synthetic peptides and ELISA analysis which indicated that 216YHPLA220 and 230DPQTSDILEA239 were the determinants of the linear B-cell epitopes recognized by 6C12 and 09980 mAbs,respectively.Furthermore,comparison of the epitopes sequences with the sequences of other REV strains indicated that the epitopes are highly conservative among other REV strains.Taken together,in these studies we have demonstrated valuable information that should be useful in clinical applications and as tool for further study of the structure and function of p27 of ALV.Also it may provide the fundamental information to development of specific serological diagnosis of REV infection,and will contribute to the rational design of vaccines by further understanding of the antigenic structure of gp90.