【摘要】 目的:构建带有Flag标签的Six1基因的慢病毒表达载体,并对表达产物Flag-Six1的生物学功能进行初步鉴定。方法:以实验室构建的真核表达载体myc-Six1为模板,根据实验需求合成引物,经PCR扩增,酶切后插入pCDHFlag载体,Western印迹检测其在人293T细胞中的表达;经Western印迹鉴定无误后,与3个包装质粒共转染293T细胞,包装成慢病毒后感染人肺癌细胞A549,经嘌呤霉素筛选2周,收集部分细胞,用Western印迹验证蛋白水平的表达,并通过细胞生长实验检测Six1对A549细胞生长的影响;提取RNA,通过qRT-PCR检测细胞中血管内皮细胞生长因子C(VEGF-C)在mRNA水平的变化。结果:构建的慢病毒表达载体pCDH-Flag-Six1能够在293T细胞中正确表达;包装成慢病毒,感染肺癌细胞A549后可正常表达;生长曲线表明Flag-Six1可促进人肺癌细胞A549的繁殖;qRTPCR结果表明Six1可升高肺癌细胞A549的VEGF-C转录水平。结论:构建了带pCDH-Flag标签的人Six1基因慢病毒表达载体,Flag-Six1能够在肺癌细胞系A549中表达,并能促进细胞增殖,这为进一步研究Six1的生物学功能奠定了重要基础。更多还原

【Abstract】 Objective:To construct the lentiviral vector of human Six1 labeled with CDH-Flag and preliminary detect the biological function of the expression product CDH-Flag-Six1. Methods:Human Six1 gene was obtained frommyc-Six1 plasmid by PCR and cloned into pCDH-Flag vector. In the human kidney 293 T cells,the expression of the constructed pCDH-Flag-Six1 was identified by Western blotting. The recombinant plasmid pCDH-FlagSix1 was co-transfected with three package plasmids into 293T cells,and then the supernatant containing lentivirus infected lung cancer A549 cell lines. After two weeks of puromycin screening,stable A549 cell lines were harvested and subjected to Western blotting,growth curve experiment and qRT-PCR to identify Six1 expression,examine growth condition,and mRNA level of VEGF-C respectively. Results:The growth curve showed Six1 overexpressing A549 cell lines accelerated proliferation. The qRT-PCR showed human Six1 gene could up-regulate the VEGF-C mRNA expression in lung cancer A549 cells. Conclusion:The lentiviral vector of pCDH-Flag-Six1 has been constructed and the CDH-Flag-Six1 protein promotes the proliferation and regulates VEGF-C expression,which has laid foundation for the further study on the function of Six1.更多还原