摘要
采用基因工程技术构建绿色荧光蛋白(GFP)报告基因质粒,有利于活细胞成像、蛋白质印迹(WB)、细胞流式分析及活细胞示踪等,从而对抗肿瘤药物进行筛选。利用聚合酶链反应(PCR)扩增NF-κB基因的启动子序列以及GFP基因片段,将其克隆到pGL6-Enhancer载体中,通过菌落PCR检测、酶切鉴定和测序证明了质粒构建成功。然后,将构建的质粒转染到HEK-293T细胞中,根据活细胞荧光成像、WB及流式细胞分析验证,构建的报告基因质粒在细胞内可正常表达。选择抗肿瘤药物雷帕霉素、紫杉醇、吉西他滨,以及本实验室正在研究开发的多肽类抗肿瘤药物M1-20、M1-21验证报告基因体系试验,发现构建的报告基因质粒可以响应药物对细胞的影响。该药物筛选平台的建立为研究抗肿瘤药物对供试细胞的影响提供了良好的试验技术体系,同时,也为构建活细胞示踪体系提供了材料。
Using genetic engineering technology, the green fluorescent protein(GFP) reporter gene plasmid was constructed,which is convenient for live-cell imaging, Western blot(WB), cell flow analysis and live-cell tracing, so as to realize the screening of anti-tumor drugs. The promoter sequence of NF-κB gene and GFP gene fragment were amplified by polymerase chain reaction(PCR), cloned into the pGL6-Enhancer vector, and the plasmid construction was proved by colony PCR, digestion identification and sequencing. The successful plasmid is then transfected into HEK-293T cells, and the reported plasmid is normally expressed within the cell based on live-cell fluorescence imaging, WB and flow cytometry. Finally, the anti-tumor drugs rapamycin, paclitaxel, gemcitabine, and the peptide anti-tumor drugs M1-20 and M1-21, which were being developed in our laboratory,were selected to verify that the gene system can respond to the effects of the drug on cells. The establishment of this drug screening platform provides a useful tool for studying the effects of anti-tumor drugs on cells, and also provides materials for building a live cell tracer system.
作者
王瑞萍
程浩杰
余雳
黄明敏
谭拥军
WANG Ruiping;CHENG Haojie;YU Li;HUANG Mingmin;TAN Yongjun(College of Biology,Hunan University,Changsha 410082,China)
出处
《激光生物学报》
CAS
2022年第6期506-511,共6页
Acta Laser Biology Sinica
基金
国家自然科学基金项目(81773169)
湖南省自然科学基金面上项目(2020JJ4180)。
关键词
报告基因
启动子
分子克隆
抗肿瘤药物
药物筛选平台
reporter genes
promoter
molecular cloning
antitumor drug
drug screening platform
