肿瘤坏死因子过表达慢病毒载体的构建及鉴定  

Construction and identification of lentivirus overexpression tumor necrosis factor-αvector

作　　者：胡霞 李伟 HU Xia;LI Wei(Department of Stomatology,Affiliated Hospital of Jinggangshan University,Ji'an,Jiangxi,343000,China;Department of Stomatology,Jinggangshan University Health Science Center,Ji'an,Jiangxi,343000,China)

机构地区：[1]井冈山大学附属医院口腔科,江西吉安343000 [2]井冈山大学医学部口腔系,江西吉安343000

出　　处：《当代医学》2023年第8期1-5,共5页Contemporary Medicine

基　　金：国家自然科学基金(81660188);江西省自然科学基金(20202BAB206037);江西省卫生厅研究项目(202130707)。

摘　　要：目的构建携带人肿瘤坏死因子-α(TNF-α)基因过表达慢病毒载体,为阐明TNF-α过表达影响牙髓干细胞(DPSCs)增殖和牙本质分化能力的研究打下基础。方法pHBLV-CMV-MCS-3×flag-EF1-ZSgreen-puro载体用EcoRI酶切,通过连接酶将TNF-α基因片段连接至线性化的载体,运用逆转录-聚合酶链式反应(RT-PCR)方法鉴定阳性克隆载体,通过转染HEK293T细胞包装病毒,并通过分析绿色荧光蛋白表达进行病毒滴度鉴定。结果成功克隆TNF-α基因片段,聚合酶链式反应(PCR)回收的TNF-α基因测序与GenBank中的序列一致,通过PCR成功扩增TNF-α基因片段并连接至病毒载体上,三质粒共转染293T细胞成功,病毒经过成功包装且其滴度为1×108 TU/L。结论成功构建携带TNF-α基因的慢病毒载体,以为后续实验中研究TNF-α对信号分子的影响提供实验依据。Objective To construct lentiviral vector carrying human tumor necrosis factor-α(TNF-α)gene,and clarify the effect of TNF-αon the ability of dental pulp stem cells(DPSCs)for proliferation and differentiation of dentin.Methods pHBLV-CMV-MCS-3×flag-EF1-ZSgreen-puro carrier digested with EcoRI enzymes,TNF-αgene segments were connected with the linearization of the carrier by ligase,the carrier of positive clones were determined by reverse transcription-polymerase chain reaction(RT-PCR),lentivirus packaged by transfection HEK293T cells,and observated lentivirus drops of green fluorescent protein expression by fluorescence microscope.Results Successfully cloned the TNF-αgene fragment,TNF-αgene sequences which recycled by polymerase chain reaction(PCR)were same with sequences in GenBank,the TNF-αwas amplificated by PCR successfully and connected to the lentivirus carrier,three plasmid transfected 293T cells successfuliy,the lentivirus packed successfuliy and the drop degree was 1×108TU/L.Conclusion Lentivirus vector with TNF-αgene was constructed successfully,and provided experimental basis on the influence of TNF-αfor signal molecules in subsequent experiments.

关 键 词：肿瘤坏死因子-Α 慢病毒 修复性牙本质 载体 

分 类 号：R78[医药卫生—口腔医学]