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目的:在真核细胞中表达hIL-17B/mFc融合蛋白并初步研究IL-17B生物学特性。方法:应用RT-PCR法克隆hIL-17B的CDS段基因序列。将测序正确的hIL-17B序列插入pCEP4质粒构建pCEP4/hIL-17B真核表达载体。转染人肾上皮293T细胞后,筛选阳性表达细胞株。RT-PCR、ELISA和Western blot等法鉴定hIL-17B分子的mRNA和蛋白水平表达。体外和体内实验验证其生物学功能。结果:成功构建了pCEP4/hIL-17B重组表达载体,并在293T细胞中稳定表达。获得的hIL-17B重组蛋白能稳定结合人单核THP-1细胞系上IL-17B受体。体外刺激THP-1,能显著促进IL-1β和TNF-α等炎症因子的分泌。体内实验发现其具有促进中性粒细胞迁移的作用。结论:稳定表达hIL-17B重组蛋白的293T细胞系的建立,为进一步研究hIL-17B的生物学功能奠定了良好的基础。
OBJECTIVE: To express hIL-17B / mFc fusion protein in eukaryotic cells and to study the biological characteristics of IL-17B. Methods: The CDS fragment of hIL-17B gene was cloned by RT-PCR. The correct sequencing of hIL-17B sequence was inserted into pCEP4 plasmid to construct pCEP4 / hIL-17B eukaryotic expression vector. 293T cells transfected with human renal epithelial cells were screened for positive expression. The expression of hIL-17B mRNA and protein was identified by RT-PCR, ELISA and Western blot. In vitro and in vivo experiments to verify its biological function. Results: The pCEP4 / hIL-17B recombinant vector was successfully constructed and stably expressed in 293T cells. The obtained hIL-17B recombinant protein stably binds to IL-17B receptor on human mononuclear THP-1 cell line. In vitro stimulation of THP-1, can significantly promote the secretion of IL-1β and TNF-α and other inflammatory cytokines. In vivo experiments found that it has the role of promoting neutrophil migration. CONCLUSION: The establishment of 293T cell line stably expressing hIL-17B recombinant protein provides a good foundation for further study on the biological function of hIL-17B.