Establishment Of Double Sandwich ELISA For Chicken Interleukin-1 And Chicken Interleukin-6 Detection

Cytokine is a kind of biological activity matter, which induced by activated immunocells and relevant cells, such as fibroblast、endothelial cell and so on, has a effect on regulating cell function. Interleukin-1（IL-1） and interleukin-6（IL-6） of chicken, excreted by mononuclear phagocytic system and T lymphocyte, exert some impacts either on non-specificity immunology regulation situation or inflammation reaction circumstance. At present, the data showed that the IL-1 and IL-6 level of chicken were undergoing obviously changes with diseases development. For example, the expression level of IL-6 in blood serum in chicken with arthritis induced by Staphylococcus aureus was increased. Experts from abroad and domestic have considered the test of IL-6 activity in blood serum of human and mouse as a important indicator to evaluate severity and resistance of the arthritis in them, but haven’t given a thought in chicken. Therefore, it is necessary to establish test method of the level of chicken IL-1 and IL-6 to help us understand them in pathogenesis and complementary diagnosis for avian diseases.In this research, we cloned and expressed IL-1 and IL-6 gene of chicken via molecular biology method, prepared antibody and established double sandwich ELISA. This study covers the following task:1 Cloned and expressed IL-1 and IL-6 gene of chickenDesigning and synthesis two pairs of specific primers according to the sequence of ChIL-1 and ChIL-6（Genebank） .Extracting total RNA from spleen cell in chicken, IL-1 and IL-6 gene were obtained by RT-PCR and were cloned into pMD18-T vector. Then recombinant plasmid was proved to be true by restriction endonuclease analysis and sequenced. By comparing with ChIL-1（NM204524） and ChIL-6（NM₂04628） published in GenBank, the homology of ChIL-1 nucleotide sequence is above 98% and that of ChIL-6 genes is 99%. The prokaryotic expression plasmid（PET-28a-ChIL-1, PGEX-KG-ChIL-6） were construced and expressed in E.coli BL21 （DE3） successfully. The expressed HIS-ChIL-1 fusion protein in E.coli BL21 （DE3） is an about 26KDa protein in the analysis of SDS-PAGE, the expressed GST-ChIL-6 fusion protein in E.coli BL21 （DE3） is 47KDa protein and confirmed by Western-blot with immunogenicity.2 Preparation and purification of poly-antibody induced by ChIL-1 and ChIL-6 proteinUsing purified recombinant protein as immunogen, Rabbit anti-HIS-ChIL-1 and anti-GST-ChIL-6 fusion protein were prepared successfully. Mouse anti-HIS-ChIL-1 and anti-GST-ChIL-6 fusion protein were prepared successfully. The depurated antibodies were obtained after these sera were purified by saturation （NH₄）₂SO₄ precipitation and Sephadex G-200 respectively.3 Primary built up double antibodies sandwich ELISA testing for ChIL-1 and ChIL-6The test method with the high specificity and sensitivity were established using mouse antibody against ChIl-1 and ChIL-6 as capturing antibody and rabbit antibody against ChIL-1 and ChIL-6 as detecting antibody. Results are following below: for the assay testing ChIL-1, the optimum concentration of coating antibody is 20ug/ml, and optimum concentration of detecting antibody is 1:1600; for the assay testing ChIL-6 , the optimum concentration of coating antibody is 50ug/ml, and optimum concentration of detecting antibody is 1:800. This assay has provided a ground work for developing double sandwich ELISA kit for detecting ChIL-1 and ChIL-6.