大鼠Slfn3基因重组腺病毒载体的构建和包装  

Constructing and Packaging Recombinant Adenovirus Vectorto Express Rat Slfn 3 Gene

作　　者：Nganguem Nzalle Yranney Brice 毛善永 莫丽莉 林慕之 张蓓[3] 周海燕[1] 王艺明[4] 况春燕[2] 刘兴德[1,5] 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)

机构地区：[1]贵州医科大学附院心血管内科,贵州贵阳550004 [2]贵州省人民医院心血管内科,贵州贵阳550002 [3]贵州医科大学附院超声科,贵州贵阳550004 [4]贵州医科大学附院心理科,贵州贵阳550004 [5]贵州中医药大学第二附属医院心血管内科,贵州贵阳550025

出　　处：《贵州医科大学学报》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]。

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.

关 键 词：大鼠 心血管疾病 Slfn3基因 重组腺病毒 构建 包装 

分 类 号：R541.1[医药卫生—心血管疾病]