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目的 :构建FHIT基因的真核表达载体及测定其在COS 1细胞中的瞬时表达。方法 :应用RT PCR方法从正常人甲状腺组织中克隆出FHIT基因 ,在KpnⅠ和 BstXⅠ两个酶切位点插入真核表达载体 pcDNA3中 ,通过酶切分析、基因测序和免疫细胞化学方法鉴定插入基因片段的正确性。结果 :FHIT基因在载体 pcDNA3中插入的位点是正确的 ,没有缺失、插入等突变 ,并在COS 1细胞中有较高的瞬时表达。结论 :成功构建了FHIT基因的真核表达载体 ,并获得其在COS 1细胞中较高的瞬时表达 ,为该基因在基因治疗中的研究提供了有力的分子工具
Objective: To construct eukaryotic expression vector of FHIT gene and determine its transient expression in COS 1 cells. METHODS: FHIT gene was cloned from normal thyroid tissue by RT-PCR and inserted into eukaryotic expression vector pcDNA3 at two restriction sites of KpnⅠand BstXⅠ. The gene was identified by enzyme digestion, gene sequencing and immunocytochemistry Fragment of the correctness. Results: The insertion site of FHIT gene in pcDNA3 was correct. There was no deletion, insertion and other mutations in FHIT gene and higher transient expression in COS 1 cells. Conclusion: The eukaryotic expression vector of FHIT gene was successfully constructed and its transient expression in COS 1 cells was successfully established, which provided a powerful molecular tool for the study of gene therapy