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1、反義RNA體外抑制丙型肝炎病毒基因表達的研究中華實驗和臨床病毒學雜志2004年l2月第l8卷4~ChineseJExpClinViral,December2004,Vol18,No.4反義RNA體外抑制丙型肝炎病毒基因表達的研究李勇年于敏吳煒強高建興王洪紀紹平王勤環(huán)斯崇文【摘要】目的研究丙型肝炎病毒(HCV)基因調(diào)控方式及反義RNA對HCV基因表達的體外抑制作用.方法采用業(yè)已建立的HCV基因調(diào)控細胞模型,以重組質(zhì)粒共轉(zhuǎn)染策略,將反義RNA重組質(zhì)粒與HCV5非翻譯區(qū)(5uTR)調(diào)控的報道基因——蟲熒光素酶
2、(1uc)重組質(zhì)粒共同轉(zhuǎn)染人肝癌細胞系(HepG2),并以不表達反義RNA的原核重組質(zhì)粒和不含有HCV5UTR的luc重組質(zhì)粒做為對照.轉(zhuǎn)染細胞經(jīng)短期培養(yǎng)后制備細胞提取液,熒光檢測法檢測luc基因的表達.結(jié)果針對HCV5uTR的反義RNA可以在體外有效抑制由HCV5uTR調(diào)控的luc基因的表達,并具有劑量依賴效應.對照重組質(zhì)粒轉(zhuǎn)染實驗證明,上述抑制作用呈序列特異性.結(jié)論HCV5uTR具有調(diào)控下游目的基因表達的重要功能,反義RNA可以在體外有效抑制由HCV5uTR介導的病毒基因的表達.【主題詞】肝炎病毒;
3、RNA,反義;基因表達調(diào)控InhibitionofhepatitisCvirusgeneexpressionbyantisensenucleotide/nvitrou~ng-nmn'.YUMin.WUWei-qiang,GAOJian-xing,ⅣGHong,JlShao-ping,ⅣGQin-huan,SIChong-wen."DepartmentofInfectiousDiseases,323HospitalofP,Xian710054,ChinaCorrespondingo7mr:LI~ng-nm
4、n,E-mail:yongnian@pub.xaonline.com【Abstract】ObjectiveTostudythemechanismofhepatitisCvirus(HCV)generegulationandtheinhibitoryeffectofantisenseRNAonHCVgeneexpressionnvitro.MethodsThehepatoblastomacellline(HepG2)wasco-transfectedbyrecombinantplasmidofantise
5、nseRNAcomplementarytoHCV5untranslatedreg"on(5UTR)andHCV5UTRDirectedluciferase(1UC)geneexpressionrecombinantplasmid.MeanwhileareversedHCV5UTRrecombinatplasnidwhichcannottranscribeasantisenseRNAinthecellandarecombinantplasmidinwhichthelUCwasregulatedbysimi
6、anvirus40(sv40)5UTRwereusedascontrolsrespectively.TheleveloflUCgeneexpressionwasdeterminedbyanenzymaticassay.ResultsTheantisenseRNAwhichdirectedtoHCV5UTRcouldobviouslyknockdowntheleveloflUCgeneexpressionandtheclose.dependentinhibitionofantisenseRNAwasobs
7、ervedatthesametime.HowevertheaboveinhibitionwasnotshowninthecellsCO.transfectedbyreversedHCV5UTRrecombinantplasmidandHCV5UTRdirectedlUCgeneexpressionrecombinantplasmid.NoreductionWSSobserredinlucgeneexpressionlevelinthecellc~transfectedbybotIlantisenseRN
8、ArecombinantplasmidandSV405UTRdirectedlUCgeneexpressionrecombinantplasmid.ConclusionHCV5Url'Rplaysanimportantroleinregulationofviralgeneexpression.TheantisenseRNAcomplementarytoHCV5UTRcouldeffectivelyinhibitthegeneexpressi
