摘要
目的:构建携带大鼠Slfn3基因的重组腺病毒表达载体,转染HEK293细胞产生病毒。方法:以p MSV-Slfn3-GFP为模板扩增Slfn3基因,再以质粒p Adeno-EF1a-MCS-MCMV-EGFP-3FLAG为载体,通过酶切、连接及转化获得p Adeno-EF1a-Slfn3-MCMV-EGFP-3FLAG重组腺病毒载体,采用内切酶Nhe I酶切及测序鉴定重组腺病毒载体,将该重组载体在HEK293细胞进行包装和放大培养,镜下观察荧光、Western blot鉴定重组质粒标签Flag蛋白在HEK293细胞中的表达。结果:构建重组腺病毒质粒p Adeno-EF1a-Slfn3-MCMV-EGFP-3FLAG,并在HEK293细胞中成功包装和扩增,经鉴定获得重组腺病毒r AD-Slfn3。结论:成功构建重组腺病毒载体p AdenoEF1-Slfn3-MCMV-EGFP-3FLAG,并在HEK293细胞成功获得重组腺病毒r AD-Slfn3。
Objective:To construct a recombinant adenovirus vectorto express rat Slfn 3 gene,andtransfect HEK293 cells to produce adenovirus expressing rat Slfn3.Methods:The Slfn 3 gene was amplified by PCR from pMSV-Slfn3-GFP.The PCR product was isolated,purified and then ligated into the plasmid pAdeno-EF1a-MCS-MCMV-EGFP-3FLAG digested with the restriction enzyme to generate recombinant pAdeno-EF1a-Slfn3-MCMV-EGFP-3FLAG,which was further transformed,amplified,isolated,digested by Nhe I and verified by sequencing.pAdeno-EF1a-Slfn3-MCMV-EGFP-3FLAGwas transfected into HEK293 cellsto produce adenovirus,which was verified by examining GFP under fluorescent microscopyand Flag expression by Western blot.Results:pAdeno-EF1a-slfn3-MCS-MCMV-EGFP-3FLAG was constructed and verified.HEK293 cells transfected with pAdeno-EF1a-slfn3-MCS-MCMV-EGFP-3FLAG produced adenovirus expressing GFP and Flag protein.Conclusion:pAdeno-EF1-Slfn3-MCMV-EGFP-3FLAG is successfully constructed and packaged with HEK293 cells to produce recombinant adenovirus expressing Slfn3.
作者
Nganguem Nzalle Yranney Brice
毛善永
莫丽莉
林慕之
张蓓
周海燕
王艺明
况春燕
刘兴德
Nganguem Nzalle Yranney Brice;MAO Shanyong;MO Lili;LIN Muzhi;ZHANG Bei;ZHOU Haiyan;WANG Yiming;KUANG Chunyan;LIU Xingde(Department of Cardiovascular Medicine,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China;Department of Cardiovascular Medicine,Guizhou Provincial People's Hospital,Guiyang 550002,Guizhou,China;Department of Ultrasound,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China;Department of Psychology,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China;Department of Cardiovascular Medicine,the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine,Guiyang 550025,Guizhou,China)
出处
《贵州医科大学学报》
CAS
2020年第3期260-264,269,共6页
Journal of Guizhou Medical University
基金
国家自然科学基金(31760294,81560056,81904319,81960315)
贵州省科技厅基金[黔科合基础(2016)1120],[黔科合平台人才(2018)5608],[黔科合平台人才(2018)5779-36],[黔科合平台人才(2018)5779-38],[黔科合平台人才(2018)5779-52]
贵阳市科技局基金[筑科合同(2017)5-14],[筑科合同(2017)30-10]
贵州省卫生计生委基金[gzwjkj(2017-1-016)]
贵州省教育厅青年科技人才成长项目[黔教合KY字(2018)182]
贵州省人社厅留学人员科技创新项目[黔人项目资助合同(2018)0003]。
关键词
大鼠
心血管疾病
Slfn3基因
重组腺病毒
构建
包装
rat
cardiovascular disease
Slfn 3 gene
recombinant adenovirus
construction
packaging
