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目的:构建真核表达载体pEGFP-claudin-1,并在293T细胞中进行表达。方法:用反转录聚合酶链反应(RT-PCR)方法扩增claudin-1开放读码框(ORF)基因,将其插入到pEGFP-C3载体的Xho I和BamH I酶切位点,构建真核表达载体pEGFP-claudin-1,酶切鉴定并测序。通过脂质体法转染293T细胞,进行荧光检测和Western blot分析。结果:构建了含有claudin-1 ORF的真核表达质粒pEGFP-claudin-1,转染293T细胞后,经荧光可见细胞膜有EGFP-claudin-1融合蛋白的表达,Western blot检测发现有相对分子质量(Mr)49000的蛋白条带。结论:成功地构建真核表达载体pEGFP-claudin-1,并在293T细胞中表达,为研究claudin-1的功能奠定了基础。
Objective: To construct eukaryotic expression vector pEGFP-claudin-1 and express it in 293T cells. Methods: The open reading frame (ORF) gene of claudin-1 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and inserted into the Xho I and BamH I restriction sites of pEGFP-C3 vector The eukaryotic expression vector pEGFP-claudin-1 was digested with restriction enzyme and sequenced. 293T cells were transfected by lipofectamine for fluorescence detection and Western blot analysis. Results: The eukaryotic expression plasmid pEGFP-claudin-1 containing claudin-1 ORF was constructed and transfected into 293T cells. The expression of EGFP-claudin-1 fusion protein was observed by fluorescence and the relative molecular mass Mr 49000 protein band. Conclusion: The eukaryotic expression vector pEGFP-claudin-1 was successfully constructed and expressed in 293T cells, which laid the foundation for the study on the function of claudin-1.