论文部分内容阅读
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。
OBJECTIVE: To construct lentivirus expression vector with Flag-tagged Six1 gene and to identify the biological function of Flag-Six1. Methods: The constructed myc-Six1 eukaryotic expression vector was used as template to synthesize the primers according to the requirements of the experiment. The PCR products were inserted into pCDHFlag vector and inserted into pCDHFlag vector. Western blotting was used to detect the expression in human 293T cells. After identification, 293T cells were co-transfected with three packaging plasmids and packaged into lentivirus to infect human lung cancer cell A549. After being selected with puromycin for 2 weeks, some cells were collected and the protein expression was verified by Western blot. Growth assays were performed to examine the effect of Six1 on the growth of A549 cells. RNA was extracted and the changes of vascular endothelial growth factor C (VEGF-C) mRNA levels were detected by qRT-PCR. Results: The constructed lentiviral vector pCDH-Flag-Six1 was correctly expressed in 293T cells. The recombinant lentivirus was packaged as a lentivirus and was expressed in A549 lung cancer cells. The growth curve showed that Flag-Six1 could promote the proliferation of human lung adenocarcinoma A549 cells. qRTPCR results show that Six1 can increase the level of VEGF-C transcription in lung cancer A549 cells. CONCLUSION: Flag-Six1, a human Six1 lentiviral vector with pCDH-Flag tag, can be expressed in lung cancer cell line A549 and can promote cell proliferation. This laid the foundation for the further study on the biological function of Six1.