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利用真核表达载体pCEP4,在人肾上皮293T细胞中表达hIL-17F/mFc融合蛋白并初步研究IL-17F生物学特性。实验应用RT-PCR法克隆hIL-17F CDS段基因序列。将测序正确的hIL-17F序列插入pCEP4质粒构建pCEP4/hIL-17F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-17F分子的mRNA和蛋白表达。体外实验验证其促炎症作用。结果表明,构建的pCEP4/hIL-17F重组表达载体,能在293T细胞中稳定表达。获得的hIL-17F重组蛋白能稳定结合Raji细胞上的IL-17RC受体。体外刺激ECV304细胞,能显著促进IL-2等炎症因子的分泌。稳定表达hIL-17F重组蛋白的293T细胞系的建立,为进一步研究hIL-17F的生物学功能奠定了良好的基础。
The eukaryotic expression vector pCEP4 was used to express hIL-17F / mFc fusion protein in human renal epithelial 293T cells and to study the biological characteristics of IL-17F. The hIL-17F CDS gene sequence was cloned by RT-PCR. The correct sequenced hIL-17F sequence was inserted into pCEP4 plasmid to construct pCEP4 / hIL-17F eukaryotic expression vector. 293T cells transfected with human renal epithelial cells were screened for positive expression. The mRNA and protein expression of hIL-17F were identified by RT-PCR, ELISA and Western blot. In vitro experiments to verify its role in proinflammatory. The results showed that the constructed pCEP4 / hIL-17F recombinant expression vector can stably express in 293T cells. The obtained hIL-17F recombinant protein stably binds to IL-17RC receptor on Raji cells. ECV304 cells stimulated in vitro, can significantly promote the secretion of IL-2 and other inflammatory cytokines. The establishment of 293T cell line stably expressing hIL-17F recombinant protein laid a good foundation for further study on the biological function of hIL-17F.