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目的初步探讨肝细胞核因子1α(HNF1α)与异染色质蛋白1γ(HP1γ)的相互作用及其功能。方法首先以成人肝cDNA文库为模板,通过聚合酶链反应(PCR)扩增出552 bp的HP1γcDNA片段,然后亚克隆到真核表达载体pcDNA3.1/Myc-HisB,转染HepG2细胞后提取总蛋白进行Western印迹鉴定,进而通过GST沉降实验验证HP1γ与HNF1α的相互作用区段,最后通过荧光素酶报告基因实验HP1γ/HNF1α相互作用的功能。结果通过测序证实真核表达载体Myc-HP1γ构建成功,Western印迹证明Myc-HP1γ可在真核细胞中表达;GST沉降结果表明HP1γ与HNF1α的N端1-189氨基酸相互作用;报告基因实验证明HP1γ抑制HNF1α的转录活性。结论 HP1γ通过与HNF1α相互作用抑制HNF1α的转录活性。
Objective To investigate the interaction and function of hepatocyte nuclear factor-1α (HNF1α) and heterochromatin 1γ (HP1γ). Methods A 552 bp fragment of HP1γ cDNA was amplified by polymerase chain reaction (PCR) from adult liver cDNA library. The fragment was subcloned into the eukaryotic expression vector pcDNA3.1 / Myc-HisB and transfected into HepG2 cells for total RNA extraction Western blotting was used to identify the interaction between HP1γ and HNF1α by GST sedimentation assay. Finally, the luciferase reporter gene was used to test the function of HP1γ / HNF1αinteraction. Results The construction of eukaryotic expression vector Myc-HP1γ was confirmed by sequencing. Western blotting showed that Myc-HP1γ was expressed in eukaryotic cells. The results of GST sedimentation showed that HP1γ interacted with 1-189 amino acids of N-terminal of HNF1α. The reporter gene was proved that HP1γ Inhibit HNF1α transcriptional activity. Conclusion HP1γ inhibits the transcriptional activity of HNF1α by interacting with HNF1α.