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.coliDH5α进行文库扩增和蓝白斑筛选,RT-PCR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cDNA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。
Objective：To isolate Mycobacterium tuberculosis H37Rv and H37Ra genes especially,and to construct two cDNA hbraries by using suppression subtractive hybridization （SSH）. Methods：We used suppression subtractive hybridization （SSH） to clone the differential genomic genes between Mycobacterium tuberculosis virulence strain H37Rv and attenuated strain H37Ra. After two times of subtractive hybridization and two times of PCR,the products of last PCR amplification were inserted into pGEM-T Easy vector and be transformed into the E. coli DH5α and screened of blue and white clones of the transformants. The subtracted cDNA library of differentially expressed genes were identiiied by RT-PCR. Results：High or especially expressed genes as tester had been obtained by SSH in correctitude reaction （H37Rv as tester） and reverse reaction （H37Ra as tester）, the eDNA libraries of A and B of Mycobacterium tuberculosis H37Rv and H37Ra were successfully constructed. 90% of the colonies were white clones, the single band of the colonies was 75% and 80%. Conclusion：The cDNA libraries of Mycobacterium tuberculosis H37Rv and H37Ra were successfully constructed using SSH technique, which lay a solid foundation for screening and cloning new specific differentially expressed genes in them.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2010年第1期61-65,共5页
Chinese Journal of Immunology
基金
国家自然科学基金资助项目(No.30872261)
关键词
分枝杆菌
结核
抑制性消减杂交
CDNA文库
Mycobacterium tuberculosis
Suppression subtractive hybridization
cDNA library
