Development of poultry immune reagent

Authors: KIM, WOOHYUN - US Department Of Agriculture (USDA); LIM, YEASEUL - US Department Of Agriculture (USDA); SUN, ZHIFENG - US Department Of Agriculture (USDA); Li, Charles; Lillehoj, Hyun

Submitted to: Research Workers in Animal Diseases Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 11/1/2018
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Objective: The objectives of this project are 1) to identify chicken immune molecules, particularly cytokines, chemokines and cell surface markers, express them as recombinant proteins, and characterize their function, and 2) to develop monoclonal antibodies (mAbs) to the target chicken molecules. Methods: Cloning of chicken genes (20 in total) for were carried out the number of sets of primers which were designed and synthesized to amplify based on the chicken genomic and mRNA sequence. The recombinant proteins were obtained by transformation into E. coli, transfection into mammalian cells, or expression in yeast in collaboration with Kingfisher Biotech. To develop mAbs against them, we immunized mice, collected lymphocytes, fused the lymphocytes with myeloma cells, screened, and generated single-cloned hybridoma. For functional characterization of the recombinant protein and mAbs, several assays have been conducted including ELISA, immunohistochemistry, Western blot, flow cytometry, qPCR, cell proliferation, and nitric oxide assay. Results: All the target we selected have shown to have critical functions in host defense against pathogens and all recombinant proteins expressed have met the quality standard for immunization in mice for mAbs production. As a progress of USAD/NIFA grant, so far, we have expressed 20 chicken proteins (11 from yeast, 9 from E. coli) and 5 proteins expressed from mammalian system for mAb development and functional study, respectively. Twenty target proteins consist of 13 cytokines (interleukin-4, 7, 10, 12p35, 12p40, 13, 16, 17F, 21, 22, 23, IFN-a, and TGF-ß), 4 chemokines (CXCLi2, CCL4, 5, and 20), 1 surface receptor, perforin and granzyme A. For mAb development, the progress is at various stages with 3 finished, 9 in characterization, 4 in production, and 2 in screening. The mAbs developed in this study represent new sets of immune reagents which are specie-specific for poultry. Conclusions: New sets of poultry immune reagents and detection methods that we have developed in this study will have a significant global impact in scientific community and society. Keywords: Immunology, Chicken, Antibody, Antigen, Cytokine