Expression Of Chicken Interleukin-4and Development Of Monoclonal Antibodies Against The Expressed Product

Interleukin-4(IL-4) is mainly secreted by Th2type cells, which has multiple immunological regulating function, such as inducing inducing Th2cells proliferation, promoting gene transcription and anti-apoptosis. IL-4plays a very important role in regulating organism humoral immune response through interacting with a variety of cell types to exert its adjusting host defences ability. The biological function of IL-4was reported in many literatures, especially in mammals. But in poultry, due to the lack of commercial tools, ChIL-4is lagging behind. In order to provide useful biological materials for immune detection, immune cells analysis and immune regulation in poultry, this study used two different ways to immunize BALB/c mice to develop monoclonal antibodies (McAbs) against ChIL-4.I. Preparation of immunogen and detecting antigenPreparation of DNA immunogen:The recombinant eukaryotic expressed plasmid containing ChIL-4was extracted from recombinant E Coli strain DH5α(pVAXl-preChIL-4) and identified with double enzyme digestion. After Agarose gel electrophoresis analysis, the corrected plasmid, pVAXl-preChIL-4was extracted based on hexadecyl trimethyl ammonium bromide (CTAB) method. The concentration of pVAXl-preChIL-4was measured as15mg/ml with good purity based on A260/A280analysis. Then pVAXl-preChIL-4was used as DNA immunogen to generate McAbs against ChIL-4.Preparation of protein immunogen:The recombinant bacteria DH5a(DE3)(pET-ChIL-4) were cultured and induced by IPTG for the expression of recombinant ChIL-4. The inclusion bodies of the recombinant bacteria were washed and purified by Ni-NTA Superflow affinity chromatography. After being renatured and concentrated, purified His-ChIL-4protein were abtained. SDS-PAGE gel electrophoresis analysis showed that ChIL-4was expressed successfully. Then the ability for B cells proliferation of recombinant ChIL-4was tested by BrdU-ELISA, the results showed that His-ChIL-4culd promote chicken B cell proliferation and the most optimal concentration of ChIL-4is40μg/ml in this proliferating assay.Preparation of detecting antigen:The recombinant bacteria BL21(pGEX-ChIL-4) were cultured and treated the same as the Preparation of protein immunogen. After purifying,recombinant ChIL-4, GST-ChIL-4was used as detecting antigen.II. Development and Characterization of Monoclonal Antibodies against Chicken Interleukin-4There we reparted two ways to develop monoclonal antibodies against ChIL-4. Firstly, plasmid pVAXl-preChIL-4was used as immunogen.4-week-old BALB/c mice were immunized with pVAXl-preChIL-4and narcotics mixture using quadriceps injection, and the mice were boosted by purified plasmid pVAXl-preChIL-4by tail vein injection.The interval of two immunizations was4weeks,4times as total with the dose as100μg/mouse each time. Secondly, His-ChIL-4protein was used as immunogen to immunize6-week-old BALB/c mice. recombinant protein emulsified in complete Freund’s adjuvant for the first immunization and in incomplete Freund’s adjuvant for the second and third injections by abdominal subcutaneous multipoint injections with the protein dose of100μg/mouse. The interval of two immunizations was2weeks. Then the mice were boosted by intravenous injection with the dose of50μg/mouse purified protein. Then the spleen cells were collected and fused with Sp2/0-Ag-14myeloma cells. Hybridoma cells were screened by indirect ELISA, the specificity of McAbs was characterized by ELISA, Dot-ELISA and Western-blot. After the characterizing of McAbs, its ability of inhibiting ChIL-4activity was tested by BrdU-ELISA proliferation assay.6hybridoma cell lines secreting mAbs against ChIL-4were obtainedand named as:3E10,4D9,5B11,5C1,5D9and5E12, respectively.The immunoglobulin subclasses of6McAbs were IgG2a, IgG1, IgG1, IgG1, IgM and IgG1, respectively, and the ELISA titers of6McAbs in cell cultural supernatant were1:800,1:12800,1:800,1:800,1:3200and1:800, and ascites were1:1.28×104,1:1.204×106,1:1.28×105,1:1.28×104,1:2.56×105and1:1.28×105, respectively. ELISA, Dot-ELISA and Western-blot results showed that6mAbs only react with ChIL-4. BrdU-ELISA proliferation assay showed that McAb5C1has the ability to inhibit ChIL-4activity of proliferate B cells. The McAbs will be very useful in the analysis of immune testing, cellular immune function, immune regulation in poultry.