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目的:构建融合基因Hyper-IL-6并在真核细胞中进行表达。方法:采用基因拼接(GeneSOEing)法将人类可溶性白细胞介素6受体和白细胞介素6的编码基因用一富含甘氨酸序列的接头经PCR扩增融合,并定向克隆至pIRES2-EGFP,构建与绿色荧光蛋白(GFP)共表达的融合重组质粒,转染哺乳动物细胞,通过绿色荧光蛋白、RT-PCR和免疫组化检测Hyper-IL-6蛋白的表达。结果:PCR扩增出1224bp的Hyper-IL-6编码序列并成功构建重组质粒pIRES-HIL-6,重组质粒转染真核细胞后经检测证实Hyper-IL-6能在真核细胞中表达。结论:人类Hyper-IL-6重组表达质粒的成功构建与真核表达为以后对其进行活性分析和生物学功能的研究提供了基础。
Objective: To construct the fusion gene Hyper-IL-6 and express it in eukaryotic cells. Methods: The coding genes of soluble human interleukin - 6 receptor and interleukin - 6 were fused by PCR with a glycine - rich linker and cloned into pIRES2 - EGFP by GeneSOEing. Green fluorescent protein (GFP) co-expression fusion recombinant plasmid was transfected into mammalian cells, and the expression of Hyper-IL-6 protein was detected by green fluorescent protein, RT-PCR and immunohistochemistry. Results: The 1224bp Hyper-IL-6 coding sequence was amplified by PCR and the recombinant plasmid pIRES-HIL-6 was successfully constructed. The recombinant plasmid was transfected into eukaryotic cells and confirmed that Hyper-IL-6 was expressed in eukaryotic cells. CONCLUSION: The successful construction and eukaryotic expression of human Hyper-IL-6 recombinant plasmids provide the basis for their subsequent activity analysis and biological functions.