Production And Characterization Of Monoclonal Antibodies With Specificity To Chicken Interleukin-2 By DNA Immunization

Chicken interleukin 2(IL-2) is an important cytokine in immune responses.The study of IL-2 is becoming hotter and hotter, especially for its expression in variable vectors. The bioassay for IL-2 mainly relies upon prestimulated T lymphoblast cells generated by concanavalin A(ConA) stimulation whereas IL-2 activity is measured by lymphocyte proliferation assay. However, this bioassay is time consuming, labor intensive, unreliable and affected by many variables including culture reagents, inhibitive factors and other growth factors . In order to quantify Chicken IL-2 generated in viro or in vitro, we need a simple, rapid and highly reliable bioassay for detection. Generation of monoclonal antibody(mAb) with specificity to Chicken IL-2 is the key step to develop the simple and rapid bioassay . We immuned BALB/c mice with chicken IL-2 cDNA in eukaryotic expression vector and screened chicken IL-2 mAb by an ELISA with chicken IL-2 products expressed in prokaryotic cells.Chicken IL-2 cDNA was cloned into eukaryotic plasmid pcDNA3.1 to construct recombinant plasmid pcDNA-IL-2. The recombinant plasmid pcDNA-IL-2 was identified by restriction enzyme digestion and PCR. Then we constructed recombinant expression vector pcDNA-GFP. It was transfected into COS-1 cell and the green fluorescence was detected by microscope. So Chicken IL-2 was cloned and expressed successfully.Chicken IL-2 cDNA was cloned into prokaryotic expression plasmid pET-30a(+)to construct recombinant plasmid pET-IL-2, which was expressed in ï¿¡.co//.BL21(DE3). The expressed product existed in the form of inclusion body. After purification, the pure product was available.The six-week-old female BALB/c mice were immunized with 1 OOug recombinant plasmid pcDNA-IL-2, once every three weeks. After the fourth immunization, spleen cells from immunized mice were fused with sp2/0-Ag-14 myeloma cells. With the chicken IL-2 product expressed in prokaryotic cells as the antigen in an indirect ELISA, three positive clones of hybridomas were obtained . The mAbs were named as 1H1K 3E12 and 4B6 respectively, and share the isotype of IgM. All mAbs had high titers in indirect ELISA.Specificity of three mAbs to chicken IL-2 were identified by indirect immunofluorescent assay(IIFA) and indirect ELISA.In the present study, recombinant vectors pcDNA-IL-2 and pET-IL-2 were constructed and expressed successfully, and three monoclonal antibodies with specificity to chicken IL-2 were obtained. This would pave the way to develop a rapid, reliable and simple assay for detecting and quantifying chicken IL-2.