Prokaryotic Expression Of Chicken Interleukin6and Development Of Monoclonal Antibody Against Chicken Interleukin6

Chicken interleukin6(ChIL-6) involves in the disease process, and plays an important role as an adjuvant in immunotherapy. It is a multifunctional cytokine, which is a current research focus of immunology. Immunoassay, bioassay and molecular bioassay are used to detect ChIL-6. Bioassay is a sensitive method and can be used to evaluated the bioactivity of ChIL-6protein, but it is a time-consuming, has a complex processing procedure, and requires a IL-6-dependent cell line. ELISA can also be used to detect ChIL-6, but antibodies against different epitopes of ChIL-6protein are required for establishment of ELISA. Therefore, a convenient, rapid and practical method for ChIL-6detection needs to be established as soon as possible. In this study, a soluble ChIL-6was obtained by prokaryotic expression, and monoclonal antibodies against ChIL-6were developed, which laid the foundation for the rapid detection of ChIL-6.A570-bp ChIL-6gene was amplified by PCR and cloned into a prokaryotic expression vector pET-32a(+). The molecular weight of the induced recombinant ChIL-6fusion protein was40kDa detected by SDS-PAGE. Most of the recombinant ChIL-6was found in the supernatant of induced bacterial lyses, indicating it is a soluble protein. The biological activity of rchIL-6protein was determined by evaluation of its ability to stimulate the proliferation of IL-6dependent7TD1cells. A concentration of10ng/mL of rchIL6could significantly stimulate the proliferation of7TD1cells. The antigenicity of the rChIL-6protein was confirmed by western-blot using a mouse anti-ChIL-6serum induced by pcDNA-IL-6plasmid as a primary antibody.8-week-old mice were immunized with pcDNA-IL-6plasmid,100μg per mouse, once two weeks. The boost of rChIL-6protein was given after forth immunization. Spleen cells of immunized mice were fused with SP2/0myeloma cells. Three monoclonal antibodies (McAbs) were screened by an indirect ELISA using the rChIL-6protein as coated antigens. These McAbs were named as1B8,4G8and6C12. The titers of ascites were1:64000,1:256000and1:256000, respectively. The isotypes of these antibodies were determined with isotyping reagents,1B8and6C12were belonged to IgM subtype,4G8belonged to IgG subtype. Indirect immunofluorescence assay showed that specific fluorescence was observed when COS-1cell transfected with pcDNA-IL-6was incubated with each McAb. Western blot showed that three McAbs revealed a specific band with rChIL-6and rChIL6-1proteins, but not rChIL6-2and His proteins. The biological activity of rchIL-6in proliferation of7TD1cells was neutralized by6C12, but not1B8an4G8.Recombinant ChIL-6was expressed in prokaryotic vector, and it had biological activity. Three McAbs against ChIL-6were obtained, and one of three McAbs had the neutralizing activity. These McAbs lay the foundation for establishment of a rapid detection method for ChIL-6and therapeutic agents.