敲低、过表达NCOA4慢病毒包装及稳转HFL1细胞株构建  

Construction of Knock-Down and Over-Express NCOA4 Lentivirus and its Stable Transgenic Human Fetal Lung Fibroblast 1

作　　者：黄德林 陈雅慧 凡志婷 石祥广[1] 黄炎 王少辉 王久存[1] 姜帅[1] HUANG Delin;CHEN Yahui;FAN Zhiting;SHI Xiangguang;HUANG Yan;WANG Shaohui;WANG Jiucun;JIANG Shuai(School of Life Sciences,Fudan University,Shanghai 200438,China)

机构地区：[1]复旦大学生命科学学院,上海200438

出　　处：《生物化工》2022年第4期1-5,共5页Biological Chemical Engineering

基　　金：国家自然科学基金青年科学基金项目(82000070);国家自然科学基金委员会面上项目(8203000633);中国博士后科学基金(2020M670982)。

摘　　要：目的:构建敲低、过表达人NCOA4基因的慢病毒载体,建立敲低、过表达NCOA4的HFL1稳转株,为研究NCOA4介导的铁死亡在肺部疾病中的作用提供一个细胞模型。方法:设计NCOA4的shRNA并插入pLKO.1载体;获取人NCOA4全基因组序列并插入pCDH载体。DNA测序验证载体目的基因序列。慢病毒包装载体共转染293T细胞,包装病毒并感染HFL1,使用Western Blot、qPCR检测其敲低、过表达效果。结果:本研究成功构建了NCOA4敲低、过表达慢病毒载体。慢病毒感染人HFL1细胞后,得到敲低、过表达NCOA4基因的HFL1稳转株。结论:成功获取了稳定敲低、过表达NCOA4的HFL1的细胞株,为研究NCOA4介导的铁死亡在肺部疾病中的作用和机制奠定了基础。Objective:To construct homo sapiens NCOA4 knock-down and over-express lentivius and establish stable transgenic lines of HFL1.In order to provide a cellular model for the mechanism of NCOA4-mediated ferroptosis in lung disease.Methods:The shRNA of NCOA4 is inserted into pLKO.1 vector and the full-length of NCOA4 cDNA is inserted into pCDH vector.The target gene sequence on the vector is verified by DNA sequencing.The lentiviral packaging vectors is then co-transfected into 293T cells to package the lentivirus and infect HFL1.The expression of NCOA4 is determined by Western Blot and qPCR.Results:The lentiviral vectors is successfully constructed.After HFL1 is infected with lentivirus,stable transgenic HFL1 strains is obtained.Conclusion:The knock-down and overexpress NCOA4 lentivirus and stable transgenic HFL1 lines are successfully constructed and lay a foundation for the mechanism of NCOA4-mediated ferroptosis in lung disease.

关 键 词：NCOA4 HFL1 慢病毒载体 铁死亡 肺部疾病 

分 类 号：Q81[生物学—生物工程]