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目的:构建含有人CXCR3基因的重组逆转录病毒载体,获得稳定表达人CXCR3分子的基因转染细胞株L929-CXCR3,研究CXCR3与其配体Mig、IP-10及I-TAC相互作用引起的L929-CXCR3的迁移效应。方法:TRIzol一步法从经PHA和IL-2活化的人PBMC中抽提总RNA,RT-PCR扩增人CXCR3全长基因,装入逆转录病毒载体pEGZ-term,与两辅助病毒载体脂质体法共转染包装细胞293T,收集培养上清感染L929细胞,500μg/mlzeocin加压筛选获得稳定表达人CXCR3分子的基因转染细胞株L929-CXCR3。采用流式细胞术和RT-PCR分别从蛋白及基因水平对CXCR3的表达进行鉴定。Transwell分析L929-CXCR3细胞在Mig、IP-10及I-TAC作用下的迁移率。结果:构建了含人CXCR3基因的重组逆转录病毒载体,建立了人CXCR3基因转染细胞株L929-CXCR3,其膜表面CXCR3分子的阳性表达率为98.4%;趋化L929-CXCR3发生迁移的最小配体浓度分别为Mig10ng/ml、IP-105ng/ml及I-TAC1ng/ml,相应迁移率分别为2.46%、2.34%及2.24%。结论:L929-CXCR3细胞株的建立为研究CXCR3信号转导与生物学功能及制备相应的单克隆抗体奠定了基础。
OBJECTIVE: To construct a recombinant retrovirus vector containing human CXCR3 gene and obtain a gene-transfected cell line L929-CXCR3 stably expressing human CXCR3. To investigate the effect of L929-CXCR3, a CXCR3 interacting with its ligand Mig, IP-10 and I- Migration effect of CXCR3. METHODS: Total RNA was extracted from human PBMCs activated by PHA and IL-2 by one-step TRIzol method. The full-length human CXCR3 gene was amplified by RT-PCR and inserted into the retroviral vector pEGZ-term, The 293T cells were co-transfected into 293T cells and the supernatant was collected to infect L929 cells. 500μg / ml zeocin was screened to obtain the gene transfection cell line L929-CXCR3 stably expressing human CXCR3. The expression of CXCR3 was identified from the protein and gene level by flow cytometry and RT-PCR respectively. Transwell analysis of the migration of L929-CXCR3 cells under the action of Mig, IP-10 and I-TAC. Results: The recombinant retroviral vector containing human CXCR3 gene was constructed and the human CXCR3 gene transfected cell line L929-CXCR3 was established. The positive expression rate of CXCR3 on the membrane surface was 98.4%. The minimal migration of chemotactic L929-CXCR3 Ligand concentrations were Mig10 ng / ml, IP-105 ng / ml and I-TAC1 ng / ml respectively, with corresponding mobilities of 2.46%, 2.34% and 2.24%, respectively. Conclusion: The establishment of L929-CXCR3 cell line lays the foundation for the study of CXCR3 signal transduction and biological function and preparation of the corresponding monoclonal antibody.