| Chicken interleukin-6 (IL-6) is an important cytokine with multiple function in immune responses, and the study of IL-6 is one of most interesting field in Immunology. Currently, the biological activity of IL-6 is mainly determined by lymphocyte proliferation assay or proliferation of cytokine-dependant cell line. However,these bioassay is time-consuming,labor-intensive,low sensitivity and affected by many variables including culture media, inhibition factors and other growth factors. Hence, a simple, rapid and highly sensitivity for chIL-6 detection need to be established. In this study, we used Chicken IL-6 expressed in eukaryotic vector as immunogen and Chicken IL-6 products expressed in prokaryotic cells as screening antigen to develop anti-chIL-6 monoclonal antibody, which may lay a basis for detection of chIL-6.Chicken IL-6 gene was cloned into eukaryotic plasmid pcDNA3.1(-) to construct recombinant plasmid pcDNA-IL-6 which was identified by restriction enzyme digestion . Then pcDNA-IL-6 was transfected into COS-1 cells by Oligofectamine. The culture supernatant was collected to detect by 7TD1 cells which growth is IL-6 dependent. The result showed that the culture supernatant could promot the growth of 7TD1 cells, indicating chIL-6 with bioactivity was generated by pcDNA-IL-6 in COS-1 cells.The chIL-6 cDNA was divided into two fragments(chIL-6-1 and chIL-6-2) and cloned into prokaryotic expression plasmid pET-32a(+) to construct recombinant plasmid pET-IL6-1 and pET-IL6-2, which were expressed induciblely in E.coli.BL21(DE3). The results of the SDS-PAGE showed that these expressed protein existed in the supernatant of cell lysate and their antigenicity were confirmed by western-blot with mouse anti-chIL-6 polyclonal antibody. After ultrasonic lysis of cells, the target proteins were purified from the supernatants by HIS-tag protein purification columns, and the concentrations of the two proteins were 1.8mg/mL and 1.0mg/mL, respectively.The eight-week-old BALB/c mice were immunized intramuscularly with 100ug recombinant plasmid pcDNA-IL-6, once each two weeks. After fifth immunization, spleen cells from immunized mice were fused with sp2/0 myeloma cells. With purified chicken IL-6 protein expressed in prokaryotic cells as coated antigens in an indirect ELISA, three positive clones of hybridomas were obtained and named as 11E8, 15B6 and 18H9, respectively. 11E8 and 15B6 were both belonged the isotype of IgG, 18H9 was belonged to the isotype of IgM.The titers of ascites with indirect ELISA were 10×2154, 10×215 and 10×211 respectively. The result of indirect immunofluorescent assay showed that all three mAbs could react specifically with COS-1 cell transfected with pcDNA-IL-6, in which 15B6 was the best one.In this study, recombinant vectors pcDNA-IL-6, pET-IL-6-1 and pET-IL-6-2 were constructed and expressed successfully, and three monoclonal antibodies with specificity to chicken IL-6 were obtained. This would pave the way to develop a rapid, reliable and simple assay for detecting and quantifying chicken IL-6.
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