Production Of Monoclonal Antibodies Of The Anti-Duck IgY Fc Fragment

The IgY antibody is one immunoglobulin molecule of ducks, there are two forms of this molecule in duck, such as intact IgY molecule and truncated IgY molecule. The purpose of this research is to produce monoclonal antibodies of anti-duck IgY Fc fragment CH3-CH4, and thus be able to detect the intact IgY of serum in duck Besides, the monoclonal antibodies of anti-duck IgY Fc fragment CH2can be able to detect the intact IgY molecule and truncated IgY molecule.1:The gene cloning and analysis of IgY Fc fragment CH2-CH3-CH4The total RNA was extracted from the spleen of cherry valley duck after the antigen stimulation of BSA, and the amplification of target gene including CHI fractional region IgY Fc fragment CH1-CH2-CH3-CH4was gained from the reaction of RT-PCR, the size of gene is1154bp.The target gene connect the T vector, and the sequencing analysis of the recombinant plasmids pMD-18T-CH1-CH2-CH3-CH4shows that the presence of the four nucleotide mutations in the gene of CH2-CH3-CH4are unintentionally mutation and the rate of nucleotide homology is99%, the rate of amino acid homology is100%. The cloning of IgY Fc fragment CH2-CH3-CH4was gained by the template of recombinant plasmid pMD-18T-CH1CH2-CH3-CH4, the size of gene is954bp.2:The prokaryotic expression of duck IgY Fc fragment CH2, CH3-CH4and CH1-CH2-CH3-CH4.The cloning of IgY Fc fragment CH2, CH3-CH4and CH1-CH2-CH3-CH4was gained by the template of recombinant plasmid pMD-18T-CH1-CH2-CH3-CH4, the size of gene are288bp,666bp and1154bp respective.Constructing prokaryotic expression vector pET-28a-CH2, pET-28a-CH3-CH4with the gene of IgY Fc fragment CH2, CH3-CH4and constructing the prokaryotic expression vector pGEX-KG-CH1-CH2-CH3-CH4with the gene of IgY Fc fragment CH1-CH2-CH3-CH4.The expression and purification of the fusion protein GST-CHI-CH2-CH3-CH4was detected by SDS-PAGE, the size of protein is48KDa and the result by western blot shows that the protein can be used to immune Balb/c mice to produce the monoclonal antibodies. The expression and purification of the fusion protein His-CH2and His-CH3-CH4is detected by SDS-PAGE, the size of protein is17KDa and26KDa.The result of western blot shows that the protein can be used to filter monoclonal antibodies.3:The production of monoclonal antibodiesThe purified fusion protein GST-CHI-CH2-CH3-CH4was used to immunize the Balb/c mice.The hybridoma strain of4F8,4H11,4A2and4A4is filtered after cell fusion which can produce the antibodies of anti-immunogen. The subtypes of4F8hybridoma strain is IgG3and the potency is up to1:1.024×106. The purified4F8monoclonal antibody can binding with the protein His-CH3-CH4at a dilution of16000times.The result of western blot of4F8monoclonal antibody shows that the intact duck IgY and chicken IgY can bind the4F8monoclonal antibody.