小鼠Atrogin-1基因真核表达载体的构建及其在293T细胞中的表达

目的：构建小鼠Atrogin-1基因真核表达载体,研究其功能,并探讨其在癌性恶液质骨骼肌萎缩中的作用机制。方法：提取小鼠C2C12细胞RNA,反转成cDNA,PCR合成含有酶切位点的Atrogin-1 cDNA全长,酶切后连接到真核表达载体pcDNA3.1,构建重组真核表达载体pcDNA3.1-Atrogin-1,经鉴定及测序证实cDNA片段大小和序列正确,然后用其转染293T细胞,Western blot法检测Atrogin-1蛋白的表达。结果：pcDNA3.1-Atrogin-1含大小、序列正确的Atrogin-1cDNA片段,转染pcDNA3.1-Atrogin-1的293T细胞裂解液中能检测到Atrogin-1蛋白高表达。结论：成功构建了Atrogin-1基因真核表达载体pcDNA3.1-Atrogin-1,并在真核细胞中表达了目的蛋白,其是研究癌性恶液质的重要工具。

Construction of Eukaryotic Expression Vector for Atrogin-1 Gene and Expression of Atrogin-1 in 293T Cells

Objective:To construct eukaryotic expression vector of mouse Atrogin-1 gene for further study on its functions in enhancing cancer cachexia-associated protein catabolic and skeletal muscle atrophy.Methods:The full length of Atrogin-1 cDNA containing two restriction sites (XhoI,EcoRI) was constructed by restriction endonuclease,then the Atrogin-1 cDNA and the digested vector pcDNA3.1 were ligated by T4 DNA ligase,and recombinant eukaryotic expression vector was formed. Its length was certificated by digestio...