ba结核分枝杆菌差异表达基因消减cdna文库

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1、万方数据型撞登笠壅旦塑剑逍遭苤塞堇鲞塑建缱堕筮蕉抚匿羞爰壹述蕉固遁壅!旦塑垒塞廛箜!塑·免疫学技术与方法·应用抑制消减杂交技术构建结核分枝杆菌差异表达基因消减cDNA文库①刘艳群杜先智(重庆医科大学第二附属医院呼吸内科，重庆400010)·61·中国图书分类号R328文献标识码A文章编号1000-4s4x{20lo)01．0061．05[摘要]目的：构建结核分枝杆菌H37Rv与H37Ra的差异表达基因消减文库，分离结核分枝杆菌差异表达cDNA片段。方法：利用抑制性消减杂交技术分析结核分枝杆菌强毒株H37Rv和弱毒株H37Ra的基因组mRNA的表达差异，并进

2、行两轮消减杂交和两次PCR，将第二次PCR产物与pGEM-T载体相连，电击转化大肠杆菌E．coliDH5a进行文库扩增和蓝白斑筛选，RT-PER鉴定差异表达文库。结果：以结核分枝杆菌强毒株H37Rv的cDNA为检测子的正相杂交和以弱毒株H37Ra的cDNA为检测子的反相杂交各自高表达或特异性表达的片段都得到选择性扩增，成功构建了差异表达cDNA文库A库和B库。所长出的菌落中90％为白色克隆，其中单一条带的克隆占75％和80％，片段大小集中在100～800bp之间。结论：利用SSH技术成功构建了结核分枝杆菌强毒株H37Rv和弱毒株H37Ra差异表达基因消减c

3、DNA文库，该消减文库的建立为进一步筛选、克隆这两种菌株之间差异表达的新基因奠定了基础。[关键词】分枝杆菌，结核；抑制性消减杂交；cDNA文库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