【作者】 汪燕燕；

【导师】 范礼斌；

【作者基本信息】 安徽医科大学 ， 遗传学， 2015， 硕士

【摘要】 目的:初步探讨E2F1蛋白和Sedlin蛋白在体内体外有无相互作用,进而为迟发性脊柱骨骺发育不良(SEDT)的靶向治疗提供新的思路。方法:以含人E2F1全长c DNA序列的质粒为模板,用常规PCR扩增E2F1,构建pc DNA3.1-E2F1-FLAG真核表达载体,将酶切鉴定正确的重组质粒测序。将pc DNA3.1-E2F1-FLAG瞬时转染至HEK 293T细胞中,同时制备GST-Sedlin融合蛋白,通过GST Pull-down技术检测E2F1蛋白与Sedlin蛋白在体外有无相互作用。将pc DNA3.1-E2F1-FLAG和p CDGFP-Sedlin分别瞬时转染到COS-7细胞中,观察两者在细胞内的定位现象,将pc DNA3.1-E2F1-FLAG与p CDGFP-Sedlin一起瞬时转染到COS-7细胞中,观察两者在细胞内的共定位现象。将pc DNA3.1-E2F1-FLAG和p CDGFP-Sedlin瞬时转染至HEK 293T细胞,通过免疫共沉淀技术检测E2F1与Sedlin蛋白在哺乳动物细胞内有无相互作用。结果:酶切鉴定和测序结果都证明了pc DNA3.1-E2F1-FLAG质粒构建成功。GST Pull-down实验表明,在体外条件下,E2F1蛋白与Sedlin蛋白存在相互作用。在荧光显微镜下观察到,E2F1蛋白主要分布在COS-7细胞核内,而Sedlin蛋白除了细胞核,在细胞质内也有分布;共定位实验观察E2F1蛋白与Sedlin蛋白在核内存在部分共定位。免疫共沉淀实验表明,在HEK 293T细胞中,E2F1蛋白与Sedlin蛋白存在相互作用。结论:在体外条件下,E2F1蛋白与Sedlin蛋白存在相互作用。在COS-7细胞中,E2F1蛋白与Sedlin蛋白在细胞核存在共定位;在HEK 293T细胞中,E2F1蛋白与Sedlin蛋白存在相互作用。更多还原

【Abstract】 Objective:The interaction between E2F1 protein and Sedlin protein in vitro and in vivo was conducted. The results would provide a new kind of insight for targeted therapy of spondyloepiphyseal dysplasia tarda(SEDT).Methods:The full-length sequence of E2F1 was amplified with conventional polymerase chain reaction(PCR).The eukaryotic expression vector pc DNA3.1-E2F1-FLAG was builded. The correct recombinant plasmid was sent to sequencing after restriction enzyme digestion. pc DNA3.1-E2F1-FLAG was transient transfected into HEK 293 T cells. At the same time GST-Sedlin fusion protein was prepared. The interaction between E2F1 protein and Sedlin protein in vitro was detected by GST Pull-down technology.In COS-7 cells,pc DNA3.1-E2F1-FLAG and p CDGFP-Sedlin were transient transfected respectively.The localization of two proteins in the cells was observed.Then the above two plasmids were transient transfected together. The co-localization of two proteins in the cells was observed. pc DNA3.1-E2F1-FLAG and p CDGFP-Sedlin were transient transfected into HEK 293 T cells. The interaction between E2F1 protein and Sedlin protein in the mammalian cells was studied using co-immunoprecipitation assay.Results:The results of enzyme digestion analysis and sequencing showed the plasmid pc DNA3.1-E2F1-FLAG was constructed successfully.GST Pull-down experiment showed the interaction between E2F1 protein and Sedlin protein in vitro was exist. Observing under the fluorescence microscope, E2F1 protein in COS-7 cells was mainly distributed in the nucleus and not distributed obvious in the cytoplasm, However, Theexpression of Sedlin protein was obsevered both in the nucleus and in the cytoplasm. The co-localization of E2F1 and Sedlin in the nucleus by co-localization experiment. Co-immunoprecipitation assay showed the interaction between E2F1 protein and Sedlin protein in HEK 293 T cells was exists.Conclusion:In vitro, the interaction between E2F1 protein and Sedlin protein was exist. In COS-7 cells, the co-localization of E2F1 and Sedlin in the nucleus. In HEK 293 T cells,the interaction between E2F1 protein and Sedlin protein was exists.更多还原