LIM结构域蛋白FHL1C抑制Notch信号途径的转录激活研究

Inhibitory Effects of Homo Sapiens Lim Protein FHL1C on Notch Signaling Transcription Activities

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 and express the full-length of human FHL1C and construct the FHL1C eukaryotic expression vec-tor and a 1entiviral expression vector.Methods：Total RNA was isolated from skeletal muscles,and the full-length FHL1C was amplified by RT-PCR wihch was ligated with pMD18T vector.After retrieve and purification,FHL1C were subcloned to pCMV-Myc by using the restricted endonuclease digestion and solutionI DNA ligase connection,the reconstructed plasmid pCMV-Myc-FHL1C was transfected into Hela and Cos7 cells,respectively.The expression was identifed by Western blot.The effect of FHL1C on Notch pathways was detected by the Dual Luciferase assay system.FHL1C was inserted into pMX-IRES-GFP to construct FHL1C-IRES-GFP,the recombi-nant plasmid pLenti6/V5-FHL1C-IRES-GFP was identified by restriction endonuclease analysis and DNA sequencing.The plasmid was packagined into 293FT Cell Line to produce lentivirus and Virus titer was measured according to the expression level of GFP.Results：The enzyme and PCR analyses revealed that the correct FHL1C was cloned.The sequence of cloned cDNA was identical to the sequence deposited in GenBank.The current expression in transfected cell was identifed by Western Blot.The FHL1C gene could downregulate the Notch signaling.The reconstructed plasmid was identified correctly by colony PCR and enzyme digestion.The production of infec-tious lenti-virus with appropriate tilters using 293FT is suitable and feasible by detect the expression of GFP.Conclusion：The eukaryotic expression vector and the recombinant lentiviral vector was constructed successfully,which will be beneficial to guiding further study on relationship between T-ALL and Notch.