Preparation and Biochemical Characteristics of a New IgG-Type Monoclonal Antibody against K Subgroup Avian Leukosis Virus

Abstract

This study focused on preparing a new IgG-type monoclonal antibody (MAb) against subgroup K avian leukosis virus (ALV-K) and identifying its biochemical characteristics. A specific gene fragment of ALV-K was amplified by polymerase chain reaction and expressed in E. coli. The purified expressed products were inoculated into BALB/c mice to prepare antibody-secreting spleen lymphocytes, and hybridoma cells were obtained after cell fusion of spleen lymphocytes and myeloma cells. A new hybridoma cell line named 30B9, which stably secreted IgG2b-antibody against ALV-K, was screened and contained 98 chromosomes. The MAb secreted by the 30B9 cells could recognize the ALV-K strain but not the ALV-A/B/J strains in an indirect immunofluorescence assay. Seventeen overlapping truncated ALV-K gp85 protein fragments were expressed, and eight peptides were artificially synthesized to analyze the MAb's antigen epitope by Western blot or enzyme-linked immunosorbent assay, and the results showed that the linear epitope was located on the 217− RRNYT −221 of ALV-K gp85 protein. A bioinformatics analysis showed that the epitope has a high antigenicity index, hydrophilicity, and surface accessibility and forms a unique linear spatial structure. Its five amino acids are highly conserved in all published ALV-K strains but are very low in ALV-A/B/J/C/D/E strains. This study provides a new biomaterial for developing specific detection methods against ALV-K.