小鼠shRNA-Slfn3重组腺病毒载体的构建及其在EPCs中转染效率的测定  

Construction of recombinant adenovirus vector with mouse shRNA-Slfn3 genes and its transfection efficiency in EPCs

作　　者：辛晨 况春燕[1,2] 刘兴德 XIN Chen;KUANG Chunyan;LIU Xingde(Department of Cardiology,the Affiliated People's Hospital of Guizhou Medical University,Guiyang 550003,Guizhou,China;Department of Cardiology,Guizhou Provincial People's Hospital,Guiyang 550003,Guizhou,China;Department of Cardiology,Guizhou University of Chinese Medicine,Guiyang 550003,Guizhou,China)

机构地区：[1]贵州医科大学附属人民医院心内科,贵州贵阳550003 [2]贵州省人民医院心内科,贵州贵阳550003 [3]贵州中医药大学心内科,贵州贵阳550003

出　　处：《贵州医科大学学报》2023年第9期1013-1019,1046,共8页Journal of Guizhou Medical University

基　　金：国家自然科学基金项目(81560056);贵州省第十二批优秀青年科技人才项目(黔科合平台人才〔2019〕5662);贵州省科技计划项目(黔科合基础〔2018〕1097);贵州省留学人员科技活动择优资助项目(黔人项目资助合同〔2018〕0003);贵州省科技平台及人才团队计划项目(黔科合平台人才2017-5405)。

摘　　要：目的构建小鼠短发夹RNA(shRNA)-Schlafen3(Slfn3)重组腺病毒载体,并检测其在内皮祖细胞(EPCs)中的转染效率。方法采用GenBank基因软件查询小鼠Slfn3基因序列,设计并合成3个siRNA片段及其引物,取腺病毒干扰载体pADV-U6-shRNA-CMV-EGFP,采用限制性内切酶进行酶切,线性化后连接siRNA片段,获得重组腺病毒载体shRNA-Slfn3,从中筛选阳性克隆抽提质粒,进行DNA测序验证;取对数生长期的人胚胎肾细胞HEK293,采用Admax系统将目的质粒转染至HEK293细胞,获得重组腺病毒shRNA-Slfn3后进行小量扩增及病毒滴度测定;取小鼠脾脏单个核细胞体外培养至对数期EPCs,用前述所得重组腺病毒shRNA-Slfn3对其进行转染48 h,采用绿色荧光蛋白量检测重组腺病毒的转染效率。结果测序验证3组目的质粒构建成功;获得shRNA-Slfn3重组腺病毒,病毒滴度分别为2.37×10^(13)pfu/L、3.16×10^(13)pfu/L及4.74×10^(13)pfu/L;EPCs转染效率为(63.64±2.58)%。结论成功构建了小鼠shRNA-Slfn3重组腺病毒载体,转染小鼠脾源EPCs后的转染效率较高。Objective To investigate the construction of recombinant adenovirus vector of mouse short hairpin RNA(shRNA)-Schlafen3(Slfn3)genes and its transfection efficiency in endothelial progenitor cells(EPCs).Method Three small interfering RNA(siRNA)fragments and their primers were designed and synthesized according to the queried mouse Slfn3 gene sequence.Adenovirus interference vector(pADV-U6-shRNA-CMV-EGFP)was linearized by restriction endonuclization,and then the recombinant adenovirus vector shRNA-Slfn3 was obtained.The positive clones were extracted and verified by DNA sequencing,while the Admax system was used to obtain recombinant adenovirus shRNA-Slfn3 by transfecting the target plasmid into HEK293 cells(Human Embryonic Kidney 293 cell),and shRNA-Slfn3 was amplified and virus titer was detected.Finally,mouse splenic EPCs were cultured in vitro and transfected with recombinant adenovirus.The transfection efficiency of the recombinant adenovirus was detected by green fluorescent protein.Results The plasmid construction functions of the three groups were verified successfully through DNA sequencing,and recombinant adenovirus shRNA-Slfn3 was obtained.The virus titers were 2.37×10^(13)pfu/L,3.16×10^(13)pfu/L,and 4.74×10^(13)pfu/L,respectively.The transfection efficiency of EPCs was(63.64±2.58)%.Conclusion The recombinant adenovirus vector of shRNA-Slfn3 can be successfully constructed and transfected into EPCs,providing a new method for the prevention and treatment of cardiovascular diseases.

关 键 词：内皮祖细胞 基因敲低技术 Slfn3基因 短发夹RNA 载体构建 包装 

分 类 号：R541.4[医药卫生—心血管疾病]