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目的在真核细胞中表达hIL-23(p19)/mFc融合蛋白并初步研究IL-23生物学特性。方法应用RT-PCR法克隆获得hIL-23(p19)/mFc基因片段。将测序正确的hIL-23(p19)/mFc序列插入pCEP4质粒构建pCEP4/hIL-23(p19)/mFc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和Western blot法鉴定hIL-23(p19)/mFc基因和蛋白表达。结果成功构建了pCEP4/hIL-23(p19)/mFc重组表达载体,并在293T细胞中稳定表达。获得的hIL-23(p19)/mFc重组蛋白能促进人T细胞hIL-17和hIL-10的表达。体外刺激巨噬细胞,能显著增加TNF-α等炎症因子的基因表达。结论成功建立稳定表达hIL-23(p19)/mFc重组蛋白的293T细胞系,可用于进一步研究hIL-23的生物学功能。
Objective To express hIL-23 (p19) / mFc fusion protein in eukaryotic cells and to study the biological characteristics of IL-23. Methods The hIL-23 (p19) / mFc gene fragment was cloned by RT-PCR. The eukaryotic expression vector pCEP4 / hIL-23 (p19) / mFc was constructed by inserting the correct sequence of hIL-23 (p19) / mFc into pCEP4 plasmid. 293T cells transfected with human renal epithelial cells were screened for positive expression. The expression of hIL-23 (p19) / mFc gene and protein was identified by RT-PCR and Western blot. Results The pCEP4 / hIL-23 (p19) / mFc recombinant expression vector was successfully constructed and stably expressed in 293T cells. The hIL-23 (p19) / mFc recombinant protein obtained can promote the expression of hIL-17 and hIL-10 in human T cells. In vitro stimulation of macrophages, can significantly increase the gene expression of inflammatory cytokines such as TNF-α. Conclusion The 293T cell line stably expressing hIL-23 (p19) / mFc recombinant protein was successfully established and could be used to further study the biological function of hIL-23.