Tet-On3G系统调控人巨细胞病毒蛋白pUL23稳定表达的人胚肺成纤维细胞(HELF)系的建立
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摘要
人巨细胞病毒(Human cytomegalovirus,HCMV)为疱疹病毒家族一员,易导致免疫力低下或缺陷的人群严重疾病,目前对HCMV编码的皮层蛋白pUL23功能的相关报道很少。本研究应用Tet-On3G诱导表达系统,在人胚肺成纤维细胞(Human embryonic lung fibroblast,HELF)建立诱导表达HCMV病毒蛋白pUL23细胞模型。将病毒基因UL23和阳性对照基因EGFP分别定向插入应答慢病毒载体pLVX-TRE3G中,获得pLVX-TRE3GUL23-3×Flag、pLVX-TRE3G-EGFP重组慢病毒载体。运用二代慢病毒包装系统与Lenti-X293T包装细胞系,制备收获慢病毒后感染人胚肺成纤维细胞HELF。遗传霉素(Geneticin,G418)和嘌呤霉素(Puromycin,Puro)抗性筛选后多西环素(Doxycycline,Dox)诱导外源基因表达。感染后的细胞在96孔板有限稀释法成单克隆,分别采用RT-PCR与Western blot技术检测病毒UL23基因在mRNA水平与蛋白水平的表达量,筛选出当诱导时高效表达且未诱导时低表达的单克隆细胞;并评估Dox诱导剂量与诱导时间对外源蛋白pUL23表达的影响。限制性内切酶与测序显示重组质粒pLVX-TRE3G-UL23-3×Flag、pLVX-TRE3G-EGFP序列和方向正确。病毒蛋白pUL23在感染细胞中能够被诱导表达;当Dox浓度高于400ng/mL时pUL23蛋白表达量不再随Dox剂量增高而增高。在Dox诱导2h后,RT-PCR结果表明在921bp处有特定条带(UL23-3×Flag基因)与此同时,Western blot实验也检测到pUL23蛋白。这些结果表明,成功构建重组慢病毒载体,包装成慢病毒感染后,病毒基因UL23能够在人胚肺成纤维细胞中诱导表达。Dox的最佳工作浓度为400ng/mL,最佳诱导时间为12h至24h之间。这将为进一步研究病毒蛋白pU23的功能奠定基础。
The human cytomegalovirus(HCMV)is a subfamily of the betaherpesvirus.It causes significant disease in immunologically compromised individuals.The function of pUL23,a tegument protein encoded by the HCMV,is poorly understood.We applied a Lenti-X Tet-On 3Gsystem to establish a flexible model in human embryonic lung fibroblasts(HELFs)in which pUL23 expression was induced.The UL23 gene of the HCMV and EGFP gene were obtained by apolymerase chain reaction(PCR).The UL23 gene and EGFP gene were inserted into the response lentiviral vector pLVX-TRE3 G,respectively,to obtain the recombinant vector pLVX-TRE3G-UL23-3×Flag and pLVX-TRE3G-EGFP.Lentiviral supernatants were generated through a second-generation packaging system using Lenti-X293 T,which was used to infect HELFs.These transduction cells screened by geneticin(G418),and puromycin(Puro)was induced by doxycycline(Dox).Transduction cells were seeded into 96-well plates to select monoclonal cells.Western blotting and RT-PCR were used to detect expression of the UL23 gene to seek cells with high expression efficiency and low background.In addition,the effect of pUL23 expression due to concentration and time of Dox induction were evaluated.The recombinant plasmids pLVX-TRE3G-UL23-3×Flag and pLVXTRE3G-EGFP were identified by DNA sequencing and restriction-enzyme digestion.In HELFs,the Dox concentration was>400ng/mL,so pUL23 expression was stable.Two hours after DOX induction,RTPCR results showed that a 921-bp-specific band(UL23-3×Flag gene)was detectable,whereas pUL23 was identified by western blotting.These results suggest that a HELF cell line stably expressing pUL23 was established with a Tet-on lentivirus expression system.The optimal concentration and time of induction was 400ng/mL and 12~24h,respectively.The established pUL23 HELF cell line could be used as a cell model to explore the new functions of viral protein pUL23.
The human cytomegalovirus(HCMV)is a subfamily of the betaherpesvirus.It causes significant disease in immunologically compromised individuals.The function of pUL23,a tegument protein encoded by the HCMV,is poorly understood.We applied a Lenti-X Tet-On 3Gsystem to establish a flexible model in human embryonic lung fibroblasts(HELFs)in which pUL23 expression was induced.The UL23 gene of the HCMV and EGFP gene were obtained by apolymerase chain reaction(PCR).The UL23 gene and EGFP gene were inserted into the response lentiviral vector pLVX-TRE3 G,respectively,to obtain the recombinant vector pLVX-TRE3G-UL23-3×Flag and pLVX-TRE3G-EGFP.Lentiviral supernatants were generated through a second-generation packaging system using Lenti-X293 T,which was used to infect HELFs.These transduction cells screened by geneticin(G418),and puromycin(Puro)was induced by doxycycline(Dox).Transduction cells were seeded into 96-well plates to select monoclonal cells.Western blotting and RT-PCR were used to detect expression of the UL23 gene to seek cells with high expression efficiency and low background.In addition,the effect of pUL23 expression due to concentration and time of Dox induction were evaluated.The recombinant plasmids pLVX-TRE3G-UL23-3×Flag and pLVXTRE3G-EGFP were identified by DNA sequencing and restriction-enzyme digestion.In HELFs,the Dox concentration was>400ng/mL,so pUL23 expression was stable.Two hours after DOX induction,RTPCR results showed that a 921-bp-specific band(UL23-3×Flag gene)was detectable,whereas pUL23 was identified by western blotting.These results suggest that a HELF cell line stably expressing pUL23 was established with a Tet-on lentivirus expression system.The optimal concentration and time of induction was 400ng/mL and 12~24h,respectively.The established pUL23 HELF cell line could be used as a cell model to explore the new functions of viral protein pUL23.
引文
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[7]Burrel S,Deback C,Agut H,Boutolleau D.Genotypic characterization of UL23thymidine kinase and UL30DNA polymerase of clinical isolates of herpes simplex virus:natural polymorphism and mutations associated with resistance to antivirals[J].Antimicrob Agents CH,2010,54(11):4833-4842.
[8]Fussenegger M.The impact of mammalian gene regulation concepts on functional genomic research,metabolic engineering,and advanced gene therapies[J].Biotecfnol Prog,2001,17(1):1-51.
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[16]Loew R,Heinz N,Hampf M,Bujard H,Gossen M.Improved Tet-responsive promoters with minimized background expression[J].BMC Biotechnol,2010,10(1):81.
[17]Park F.Lentiviral vectors:are they the future of animal transgenesis?[J].Physiol Genomics,2007,31(2):159-173.
[18]Tiscornia G,Singer O,Verma I M.Production and purification of lentiviral vectors[J].Nat Protoc,2006,1(1):241-245.
[19]Wickham H.ggplot2:Elegant Graphics for Data Analysis[M].Springer Publishing Company,Incorporated,2016,16(2):180-185.
[20]Benjamini Y,Yekutieli D.The control of the false discovery rate in multiple testing under dependency[M].Ann Stat,2001,29(4):1165-1188.
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[22]Fan X,Petitt M,Gamboa M,Huang M,Dhal S,Druzin M L,Wu J C,Chen-Tsai Y,Nayak N R.Transient,inducible,placenta-specific gene expression in mice[J].Endocrinology,2012,153(11):5637-5644.
[23]王晓,朱君,吴亮,吴腊梅,刘原,苏丹华,等.Tet-on慢病毒系统表达弓形虫ROP18的研究[J].中国寄生虫学与寄生虫病杂志,2015,33(1):25-28.
[24]Gompels U A,Nicholas J,Lawrence G,Jones M,Thomson B J,Martin M E D,Efstathiou S,Craxton M,Macaulay H A.The DNA Sequence of human herpesvirus-6:structure,coding content,and genome evolution[J].Virology,1995,209(1):29-51.
[25]Megaw A G,Rapaport D,Avidor B,Frenkel N,Davison A J.The DNA sequence of the RK strain of human herpesvirus 7[J].Virology,1998,244(1):119-132.
[26]Nicholas J.Determination and analysis of the complete nucleotide sequence of human herpesvirus[J].J Virol,1996,70(9):5975-5989.
[27]Rawlinson W D,Farrell H E,Barrell B G.Analysis of the complete DNA sequence of murine cytomegalovirus[J].J Virol,1996,70(12):8833.
[28]Vink C,Beuken E,Bruggeman C A.Complete DNAsequence of the rat cytomegalovirus genome[J].J Virol,2000,74(16):7656.
[29]Bahr U,Darai G.Analysis and characterization of the complete genome of tupaia(tree shrew)herpesvirus[J].J Virol,2001,75(10):4854-4870.
[30]Loew R,Heinz N,Hampf M,Bujard H,Gossen M.Improved Tet-responsive promoters with minimized background expression[J].BMC Biotechnol,2010,10(1):81.
[31]Fife R S,Jr S G,Roth B J,Proctor C.Effects of doxycycline on human prostate cancer cells in vitro[J].Cancer Lett,1998,127(2):37-41.
[2]Mocarski E,Rossetto C C,Tarrant-Elorza M,Pari G S.Cis and trans acting factors involved in human cytomegalovirus experimental and natural latent infection of CD14(+)monocytes and CD34(+)cells[J/OL].PLoS Pathog,2013,9(5):e1003366.
[3]Landolfo S,Gariglio M,Gribaudo G,Lembo D.The human cytomegalovirus[J].Aliment Pharmacol Ther,2003;98(3):269-297.
[4]Dunn W,Chou C,Li H,Hai R,Patterson D,Stolc V,Zhu H,Liu F.Functional profiling of a human cytomegalovirus genome[J].PNAS,2003,100(24):14223-14228.
[5]Lv Y L,An Z L,Chen H,Wang Z H,Liu L H.Mechanism of curcumin resistance to human cytomegalovirus in HELF cells[J].BMC Complem Altern M,2014,14(1):284.
[6]Yu X,Shah S,Lee M,Dai W,Lo P,Britt W,Zhu H,Liu F,Zhou Z H.Biochemical and structural characterization of the capsid-bound tegument proteins of human cytomegalovirus[J].J Struct B,2011,174(3):451-460.
[7]Burrel S,Deback C,Agut H,Boutolleau D.Genotypic characterization of UL23thymidine kinase and UL30DNA polymerase of clinical isolates of herpes simplex virus:natural polymorphism and mutations associated with resistance to antivirals[J].Antimicrob Agents CH,2010,54(11):4833-4842.
[8]Fussenegger M.The impact of mammalian gene regulation concepts on functional genomic research,metabolic engineering,and advanced gene therapies[J].Biotecfnol Prog,2001,17(1):1-51.
[9]廖伟.一种新型的可诱导性基因表达系统:Tet—off/Tet—on基因表达系统[J].医学分子生物学杂志,1998(3),97-100.
[10]Berens C,Hillen W.Gene regulation by tetracyclines.Constraints of resistance regulation in bacteria shape TetR for application in eukaryotes[J].Eur J Biochem,2003,270(270):3109-3121.
[11]Gossen M,Bujard H.Tight control of gene expression in mammalian cells by tetracycline-responsive promoters[J].PNAS,1992,89(12):5547.
[12]Zhou X,Vink M,Klaver B,Berkhout B,Das AT.Optimization of the Tet-On system for regulated gene expression through viral evolution[J].Gene Ther,2006,13(19):1382-1390.
[13]高杰,严会文,李红,黄悦,胡蓉,苏敏.β-神经生长因子基因克隆和诱导表达及其对PC12细胞的诱导分化作用[J].解剖学报,2016,47(4):476-481.
[14]Mertens J,Stüber K,Poppe D,Doerr J,Ladewig J,Oliver B,Koch P.Embryonic stem cell–based modeling of tau pathology in human neurons[J].Am J Pathol,2013,182(5):1769-1779.
[15]孙勇.慢病毒系统HBV基因可调控表达动物模型的构建[D].复旦大学,2012.
[16]Loew R,Heinz N,Hampf M,Bujard H,Gossen M.Improved Tet-responsive promoters with minimized background expression[J].BMC Biotechnol,2010,10(1):81.
[17]Park F.Lentiviral vectors:are they the future of animal transgenesis?[J].Physiol Genomics,2007,31(2):159-173.
[18]Tiscornia G,Singer O,Verma I M.Production and purification of lentiviral vectors[J].Nat Protoc,2006,1(1):241-245.
[19]Wickham H.ggplot2:Elegant Graphics for Data Analysis[M].Springer Publishing Company,Incorporated,2016,16(2):180-185.
[20]Benjamini Y,Yekutieli D.The control of the false discovery rate in multiple testing under dependency[M].Ann Stat,2001,29(4):1165-1188.
[21]Patterson G H,Knobel S M,Sharif W D,Kain S R,Piston D W.Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy[J].Biophys J,1997,73(5):2782-2790.
[22]Fan X,Petitt M,Gamboa M,Huang M,Dhal S,Druzin M L,Wu J C,Chen-Tsai Y,Nayak N R.Transient,inducible,placenta-specific gene expression in mice[J].Endocrinology,2012,153(11):5637-5644.
[23]王晓,朱君,吴亮,吴腊梅,刘原,苏丹华,等.Tet-on慢病毒系统表达弓形虫ROP18的研究[J].中国寄生虫学与寄生虫病杂志,2015,33(1):25-28.
[24]Gompels U A,Nicholas J,Lawrence G,Jones M,Thomson B J,Martin M E D,Efstathiou S,Craxton M,Macaulay H A.The DNA Sequence of human herpesvirus-6:structure,coding content,and genome evolution[J].Virology,1995,209(1):29-51.
[25]Megaw A G,Rapaport D,Avidor B,Frenkel N,Davison A J.The DNA sequence of the RK strain of human herpesvirus 7[J].Virology,1998,244(1):119-132.
[26]Nicholas J.Determination and analysis of the complete nucleotide sequence of human herpesvirus[J].J Virol,1996,70(9):5975-5989.
[27]Rawlinson W D,Farrell H E,Barrell B G.Analysis of the complete DNA sequence of murine cytomegalovirus[J].J Virol,1996,70(12):8833.
[28]Vink C,Beuken E,Bruggeman C A.Complete DNAsequence of the rat cytomegalovirus genome[J].J Virol,2000,74(16):7656.
[29]Bahr U,Darai G.Analysis and characterization of the complete genome of tupaia(tree shrew)herpesvirus[J].J Virol,2001,75(10):4854-4870.
[30]Loew R,Heinz N,Hampf M,Bujard H,Gossen M.Improved Tet-responsive promoters with minimized background expression[J].BMC Biotechnol,2010,10(1):81.
[31]Fife R S,Jr S G,Roth B J,Proctor C.Effects of doxycycline on human prostate cancer cells in vitro[J].Cancer Lett,1998,127(2):37-41.