反义RNA体外抑制丙型肝炎病毒基因表达的研究.docVIP

反义RNA体外抑制丙型肝炎病毒基因表达的研究 中华实验和临床病毒学杂志2004年l2月第l8卷4~ChineseJExpClinViral,December2004,Vol18,No.4 反义RNA体外抑制丙型肝炎病毒 基因表达的研究 李勇年于敏吴炜强高建兴王洪纪绍平王勤环斯崇文 【摘要】目的研究丙型肝炎病毒(HCV)基因调控方式及反义RNA对HCV基因表达的体外抑 制作用.方法采用业已建立的HCV基因调控细胞模型,以重组质粒共转染策略,将反义RNA重组 质粒与HCV5非翻译区(5uTR)调控的报道基因——虫荧光素酶(1uc)重组质粒共同转染人肝癌细 胞系(HepG2),并以不表达反义RNA的原核重组质粒和不含有HCV5UTR的luc重组质粒做为对照. 转染细胞经短期培养后制备细胞提取液,荧光检测法检测luc基因的表达.结果针对HCV5uTR 的反义RNA可以在体外有效抑制由HCV5uTR调控的luc基因的表达,并具有剂量依赖效应.对照 重组质粒转染实验证明,上述抑制作用呈序列特异性.结论HCV5uTR具有调控下游目的基因表 达的重要功能,反义RNA可以在体外有效抑制由HCV5uTR介导的病毒基因的表达. 【主题词】肝炎病毒;RNA,反义;基因表达调控 InhibitionofhepatitisCvirusgeneexpressionbyantisensenucleotide/nvitrou~ng-nmn.YUMin.WU Wei-qiang,GAOJian-xing,ⅣGHong,JlShao-ping,ⅣGQin-huan,SIChong-wen.Departmentof InfectiousDiseases,323HospitalofP,Xian710054,China Correspondingo7mr:LI~ng-nmn,E-mail:yongnian@ 【Abstract】ObjectiveTostudythemechanismofhepatitisCvirus(HCV)generegulationandthe inhibitoryeffectofantisenseRNAonHCVgeneexpressionnvitro.MethodsThehepatoblastomacellline (HepG2)wasco-transfectedbyrecombinantplasmidofantisenseRNAcomplementarytoHCV5untranslated regon(5UTR)andHCV5UTRDirectedluciferase(1UC)geneexpressionrecombinantplasmid.Meanwhilea reversedHCV5UTRrecombinatplasnidwhichcannottranscribeasantisenseRNAinthecellandarecombinant plasmidinwhichthelUCwasregulatedbysimianvirus40(sv40)5UTRwereusedascontrolsrespectively.The leveloflUCgeneexpressionwasdeterminedbyanenzymaticassay.ResultsTheantisenseRNAwhichdirectedto HCV5UTRcouldobviouslyknockdowntheleveloflUCgeneexpressionandtheclose.dependentinhibitionof antisenseRNAwasobservedatthesametime.HowevertheaboveinhibitionwasnotshowninthecellsCO. transfectedbyreversedHCV5UTRrecombinantplasmidandHCV5UTRdirectedlUCgeneexpression recombinantplasmid.NoreductionWSSobserredinlucgeneexpressionlevelinthecellc~transfectedbybotIl antisenseRNArecombinantplasmidandSV405UTRdirectedlUCgeneexpressionrecombinantplasmid. ConclusionHCV5UrlRplaysanimportantroleinregulationofviralgeneexpression.TheantisenseRNA complementarytoHCV5UTRcouldeffectivelyinhibittheg