凝血因子Ⅸ无义突变真核表达载体的构建、鉴定及其表达的开题报告.docxVIP

凝血因子Ⅸ无义突变真核表达载体的构建、鉴定及其表达的开题报告 摘要： 凝血因子IX（FIX）是人体内一种重要的凝血因子，参与凝血酶生成、凝血系统的稳定和纤溶酶系统的调节，其严重缺乏会导致血友病B。本文研究了FIX基因的无义突变和真核表达载体的构建、鉴定及其表达。通过PCR扩增和测序，得到人FIX基因的无义突变，并在基因序列中引入限制性内切酶切位点。随后，利用限制性内切酶切割和连接技术，将该基因放入真核表达载体中，并通过限制酶切割和测序鉴定其正确性。最后，通过转染等技术将载体转入哺乳动物细胞中，并利用Western blot技术鉴定其表达性。 关键词：凝血因子IX；无义突变；真核表达载体；转染；Western blot Abstract: Coagulation factor IX (FIX) is an important coagulation factor in the human body, which is involved in thrombin generation, stabilization of the coagulation system, and regulation of the fibrinolysis system. Severe deficiency of FIX can lead to hemophilia B. In this study, we investigated the nonsense mutation of the FIX gene, constructed and identified a eukaryotic expression vector, and expressed it. By PCR amplification and sequencing, the nonsense mutation of the human FIX gene was obtained, and a restriction endonuclease cleavage site was introduced into the gene sequence. Subsequently, the gene was inserted into the eukaryotic expression vector using restriction endonuclease cleavage and ligation techniques, and the accuracy of the vector was verified by restriction enzyme cleavage and sequencing. Finally, the vector was transfected into mammalian cells using transfection techniques, and its expression was identified by Western blot. Keywords: coagulation factor IX; nonsense mutation; eukaryotic expression vector; transfection; Western blot