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目的:克隆人表达FHL1C基因以及建立真核表达载体和慢病毒载体。方法:从人的骨骼肌细胞来源的cDNA中扩增并克隆人FHL1C基因的编码区,连接至pMD-18T载体酶切鉴定后测序。序列测定确认后,双酶切pMD18T-FHL1C回收片段,插入真核表达载体,构建真核表达载体pCMV-Myc-FHL1C酶切鉴定正确后,转染Hela细胞及Cos7细胞,用Western Blot检测其在转染细胞中的表达,用双荧光素报告基因系统检测其对Notch信号作用。构建FHL1C-IRES-GFP表达单元,建立慢病毒表达载体plen-ti6/V5-FHL1C-IRES-GFP,包装慢病毒后感染培养的细胞,用免疫荧光显微镜、Western Blot检测分析其在被感染的细胞中的表达。结果:通过PCR方法成功扩增了人FHL1C基因的编码区。通过转染Hela及Cos7细胞,使用Western Blot检测其蛋白水平表达,双荧光报告基因系统分析均能够下调激活的Notch信号。成功构建了FHL1C慢病毒表达载体pLenti6/V5-FHL1C-IRES-GFP,包装慢病毒,把获取的慢病毒感染细胞后通过荧光显微镜证实被感染的细胞绿色荧光蛋白正确表达,Western Blot检测证实其表达。结论:成功建立起FHL1C真核表达载体及慢病毒表达系统,为研究急性T淋巴细胞白血病与Notch信号转导通路之间的关系奠定了基础。
Objective: To clone human FHL1C gene and construct eukaryotic expression vector and lentiviral vector. METHODS: The coding region of human FHL1C gene was amplified from human skeletal muscle cell-derived cDNA and ligated into pMD-18T vector for sequencing. After confirmed by sequencing, the recombinant plasmid pMD18T-FHL1C was digested with restriction endonucleases and inserted into the eukaryotic expression vector to construct the eukaryotic expression vector pCMV-Myc-FHL1C. The recombinant plasmid pCMV-Myc-FHL1C was identified by restriction analysis and transfected into Hela cells and Cos7 cells. Western Blot The expression in transfected cells was measured using the dual fluorescein reporter gene system for Notch signaling. The lentiviral expression vector plen-ti6 / V5-FHL1C-IRES-GFP was constructed and the infected cells were infected with lentivirus. The infected cells were infected with lentivirus by immunofluorescence microscopy and Western Blot. In the expression. Results: The coding region of human FHL1C gene was successfully amplified by PCR. Transfection of Hela and Cos7 cells by Western Blot detection of protein expression, dual fluorescent reporter gene system analysis can down-regulate the activation of Notch signal. The recombinant lentiviral vector pLenti6 / V5-FHL1C-IRES-GFP was successfully constructed and packaged with lentivirus. The infected lentivirus was transfected into the lentiviral vector to confirm the expression of green fluorescent protein (GFP) in infected cells by fluorescence microscopy. Western Blot . Conclusion: The eukaryotic expression vector of FHL1C and lentivirus expression system were successfully established, which laid the foundation for the study of the relationship between Notch signal transduction pathway and acute lymphoblastic leukemia.