摘要
构建具有EGFP标签的抑癌基因p53表达载体,并验证其对肺癌细胞A549的影响。以质粒pcDNA3.1-p53为模板,设计引物并进行PCR扩增获得目的基因p53,将获得的目的基因克隆至载体pcDNA3.1-EGFP上,用琼脂糖凝胶电泳检测目的条带,双酶切以及测序验证。将克隆好的载体,利用脂质体法转染A549细胞,荧光显微镜下观察,同时用MTT法测不同时间段细胞OD值。结果表明,琼脂糖凝胶电泳结果显示,目的基因扩增成功。双酶切及DNA测序结果均证实,成功构建了载体pcDNA3.1-EGFP-p53。转染A549细胞后,荧光显微镜下观察到绿色荧光。MTT法测定结果表明,目的基因转染的A549细胞组所测得的OD值明显低于对照组(p<0.01)。因此,具有EGFP标签的抑癌基因p53表达载体pcDNA3.1-EGFP-p53构建成功,转染A549细胞后,对A549细胞具有一定抑制作用。为后续继续研究p53-MDM2生物发光能量共振转移作用奠定了基础。
The EGFP-tagged tumor suppressor gene p53 expression vector was constructed and its effect on lung cancer cell A549 was verified.Primers were designed for PCR amplification to obtain the target gene p53using the plasmid pcDNA3.1-p53 as template.The obtained target gene was cloned into the vector pcDNA3.1-EGFP, the target band and its double digestion fragment was detected by agarose gel electrophoresis, then verified by DNA sequencing.The cloned vector was transfected into A549 cells by liposome method, observed under a fluorescence microscope, and the OD value of cells was measured by MTT method at different time points.The results of agarose gel electrophoresis showed that the target gene was amplified successfully. The results of double enzyme digestion and DNA sequencing confirmed that the vector pcDNA3.1-EGFP-p53 was constructed successfully.Green fluorescence was observed in A549 cells under a fluorescence microscope after transfection.The results of MTT assay showed that the OD value measured by the target gene transfected A549 cell group was significantly lower than that of the control group (p<0.01).Therefore,the EGFP-tagged tumor suppressor gene p53 expression vector pcDNA3.1-EGFP-p53 was constructedsuccessfully, and it has a certain inhibitory effect on A549 cells after transfection..It provides a basis for the subsequent study on bioluminescence energy resonance transfer of p53-MDM2.
作者
胡昌亮
尹志刚
俸婷婷
周英
HU Chang-liang;YIN zhi-gang;FENG Ting-ting;ZHOU Ying(College of Life Sciences, Guizhou University, Guiyang , Guizhou 550025, China;College of Pharmacy, Guiyang College of Traditional Chinese Medicine,Guiyang,Guizhou 550002;Guizhou Provincial Research and Development Center for Medicinal and Edible Plant Resources, Guiyang, Guizhou 550025,China;GuizhouProvincialLab for Applying and Developmentof Medicinal and EdiblePlant Resources Guiyang ,Guizhou 550025,China.)
出处
《山地农业生物学报》
2019年第1期1-5,共5页
Journal of Mountain Agriculture and Biology
基金
国家自然科学基金地区基金(81660576)
贵州省科技计划社会攻关计划项目(黔科合【2016】支撑2909)
贵州省科技重大专项子课题(黔科合J重大字[2015]2002-3-4)
贵州省高层次创新型人才培养(黔科合人才(2015)4032号)
贵州省药食同源植物资源开发工程技术研究中心(黔科合G字(2015)4001号)
