应用抑制消减杂交技术构建耐多药结核杆菌差异表达基因消减cDNA文库

Construction of subtracted cDNA library of diferentially expressed genes of multidrug resistant tubercle bacillus by suppression subtracted hybridization

目的:构建结核杆菌耐多药株与敏感株的差异表达消减cDNA文库,进一步探索结核杆菌耐多药的分子机制。方法:以耐多药菌株cDNA为实验组(Tester),敏感株cDNA为驱动组(Driver)应用抑制消减杂交(Suppression subtractive hybridiza-tion,SSH)技术结合T/A克隆技术构建结核杆菌耐多药株与敏感株的差异表达消减cDNA文库。结果:成功构建了耐多药结核菌株差异表达消减cDNA文库,获得113个差异表达cDNA片段。结论:研究表明SSH技术是筛选新功能基因的有效方法;多种已知或未知基因均参与了结核杆菌耐多药的调节,大规模筛选与克隆这些基因为进一步研究结核杆菌耐多药机制的产生奠定了必要的理论基础。

Objective： To build the subtracted cDNA library of differentially expressed genes of multidrug-resistant tubercle bacillus （MDR-TB）and to further discuss the molecular mechanism of MDR-TB. Methods：Tester was MDR-TB and Driver was sensitive tu- berculosis. Suppression subtractive hybridization（SSH） and T/A cloning technology were done to build the subtracted cDNA library of differentially expressed genes of MDR-TB. Results：The subtracted cDNA library of differentially expressed genes of MDR-TB was successfully built and 113 differentially expressed cDNA fragements of MDR-TB were obtained. Sequencing and homology analysis showed that 5 of them were novel cDNA sequences and 5 sequenced genes were reported to be related with MDR in TB. Conclusions： SSH is an effective method for screening new function genes. Many genes both known and unknown are in correlation with MDR in TB. Discovery of these genes provides a solid foundation for the explanation of MDR mechanism in TB.