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G蛋白偶联受体84(GPR84)是C9-C14的中链脂肪酸受体,不仅在脂肪酸代谢和机体免疫反应中发挥着重要作用,而且与多发性硬化症、内毒素血症等炎症性免疫疾病有着密切联系.因此,找到以GPR84为靶点的配体,可能为治疗这些疾病提供一种有效的方法.构建稳定过表达GPR84受体蛋白的细胞系,为筛选GPR84为靶点的配体和研究这些免疫疾病提供了重要途径.本文将含GPR84和Gα16基因质粒共转染到HEK293细胞中,经过抗生素抗性筛选,挑取稳定表达GPR84蛋白的HEK293细胞系.用RT-PCR、免疫荧光染色等实验方法检测了GPR84基因和其编码受体蛋白的表达;用钙流实验、环腺苷酸实验、蛋白质印迹分析、流式细胞分析证实了表达的GPR84具有完整的生物学活性,并利用该细胞系进行了药物高通量筛选,得到了新的GPR84拮抗剂,为进一步阐明GPR84的生物学功能,研究并治疗与其相关免疫疾病奠定了基础.
G protein-coupled receptor 84 (GPR84), a medium-chain fatty acid receptor of C9-C14, plays an important role not only in fatty acid metabolism and in the immune response of the body, but also with inflammatory diseases such as multiple sclerosis and endotoxemia Therefore, to find GPR84-targeted ligands may provide an effective method for the treatment of these diseases.Constructing a cell line stably overexpressing the GPR84 receptor protein, a ligand for screening GPR84 as a target And provide an important way to study these immune diseases.In this paper, the plasmids containing GPR84 and Gα16 were cotransfected into HEK293 cells, and the HEK293 cell line stably expressing GPR84 protein was selected by antibiotic resistance screening.Using RT-PCR, immunofluorescence Staining and other methods to detect GPR84 gene and its receptor protein expression; calcium flow experiments, cyclic adenosine acid assay, Western blot analysis, flow cytometry analysis confirmed the expression of GPR84 has complete biological activity, and use The cell line was screened by high-throughput drug, and a new GPR84 antagonist was obtained, which laid the foundation for further elucidating the biological function of GPR84 and studying and treating the related immune diseases .