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1、萬方數(shù)據(jù)型撞登笠壅旦塑劍逍遭苤塞堇鲞塑建繾墮筮蕉撫匿羞爰壹述蕉固遁壅!旦塑壘塞廛箜!塑·免疫學技術(shù)與方法·應(yīng)用抑制消減雜交技術(shù)構(gòu)建結(jié)核分枝桿菌差異表達基因消減cDNA文庫①劉艷群杜先智(重慶醫(yī)科大學第二附屬醫(yī)院呼吸內(nèi)科，重慶400010)·61·中國圖書分類號R328文獻標識碼A文章編號1000-4s4x{20lo)01．0061．05[摘要]目的：構(gòu)建結(jié)核分枝桿菌H37Rv與H37Ra的差異表達基因消減文庫，分離結(jié)核分枝桿菌差異表達cDNA片段。方法：利用抑制性消減雜交技術(shù)分析結(jié)核分枝桿菌強毒株H37Rv和弱毒株H37Ra的基因組mRNA的表達差異，并進
2、行兩輪消減雜交和兩次PCR，將第二次PCR產(chǎn)物與pGEM-T載體相連，電擊轉(zhuǎn)化大腸桿菌E．coliDH5a進行文庫擴增和藍白斑篩選，RT-PER鑒定差異表達文庫。結(jié)果：以結(jié)核分枝桿菌強毒株H37Rv的cDNA為檢測子的正相雜交和以弱毒株H37Ra的cDNA為檢測子的反相雜交各自高表達或特異性表達的片段都得到選擇性擴增，成功構(gòu)建了差異表達cDNA文庫A庫和B庫。所長出的菌落中90％為白色克隆，其中單一條帶的克隆占75％和80％，片段大小集中在100～800bp之間。結(jié)論：利用SSH技術(shù)成功構(gòu)建了結(jié)核分枝桿菌強毒株H37Rv和弱毒株H37Ra差異表達基因消減c
3、DNA文庫，該消減文庫的建立為進一步篩選、克隆這兩種菌株之間差異表達的新基因奠定了基礎(chǔ)。[關(guān)鍵詞】分枝桿菌，結(jié)核；抑制性消減雜交；cDNA文庫ConstructionofsubtractedcDNAlibrarybysuppressionsubtractedhybridizationfordifferentiallyexpressedgenesinMycobacteriumtuberculosisⅡuYo／-t-Q／／／t，DUXian·Zhi．DepartmentofRespiratoryMedicine，theSecondAffiliatedHospi
4、tal，ChongqingMedicalUni—versity，Chongqing400010，China[Abstract]Objective：ToisolateMycobaeteriumtuberculosisH37RvandH37Ragenesespecially，andtoconstructtwocDNAlibrariesbyusingsuppressionsubtraefivehybridization(SSH)．Methods：Weusedsuppressionsubtraetivehybridization(SSH)toclonethedif
5、ferentialgenomicgenesbetweenMyeobacteriumtuberculosisvirulencestrainH37RvandattenuatedstrainH37Ra．Aftertwotimesofsubtraetivehy·bfidizationandtwotimesofPCR，theproductsoflastPCRamplificationwereinsertedintopGEM-TEasyvectorandbetransformedintotheE．eoliDH5aandscreenedofblueandwhiteclo
6、nesofthetransformants．ThesubtractedcDNAlibraryofdifferentiallyexpressedgeneswerei—denfifiedbyRT-PCR．Results：HJ【ghorespeciallyexpressedgenesastesterhadbeenobtainedbySSHineorrectitudereaction(H37Rvastester)andreversereaction(H37Raastester)，thecDNAlibrariesofAandBofMyeobaeteriumtuber
7、culosisH37RvandH37RawereSUCCESS—fullyconstructed．90％ofthecolonieswerewhiteclones，thesinglebandofthecoloniesWas75％and80％．Conclusion：ThecDNAlibrariesofMycobacteriumtuberculosisH37RvandH37RaweresuccessfullyconstructedusingSSHtechnique，whichlayasolidfoundationforscreeningandcloningnew
8、specificdifferentiallyexpressedge
