结核分枝杆菌休眠期和活跃期差异表达基因分析

Analysis of differentially expressed genes between dormant and active Mycobacterium tuberculosis

目的探讨结核分枝杆菌休眠的机制,寻找结核分枝杆菌休眠期高表达基因。方法分别取对数生长期结核分枝杆菌和已培养100 d的陈旧结核分枝杆菌(荧光染色证实为休眠菌),提取基因组RNA,DNA酶处理后用RNA回收试剂盒回收RNA,用mRNA纯化试剂盒纯化RNA,利用抑制性消减杂交(SSH)技术分析两时期结核分枝杆菌的基因组mRNA的表达差异;通过基因克隆、基因测序和序列分析,寻找差异表达基因;采用实时荧光定量PCR鉴定高表达基因。结果通过SSH杂交,以休眠结核分枝杆菌cDNA为检测子的正相杂交和以对数生长期结核分枝杆菌cDNA为检测子的反相杂交的各自检测子高表达或特异性表达的片段都得到了选择性扩增,杂交产物经基因克隆、转化、测序分析以及用BioEdit软件比对和Blast分析,正相得到14个休眠结核分枝杆菌特异性表达或高表达的功能基因,反相得到17个对数生长期结核分枝杆菌特异性表达或高表达的功能基因。结论利用SSH杂交技术筛选到休眠结核分枝杆菌和对数生长期结核分枝杆菌的差异表达基因,为休眠结核分枝杆菌休眠机制的研究奠定了基础。

Objective To examined the mechanism for the dormancy of Mycobacterium tuberculosis and to screen for key genes of dormant M.tuberculosis.Methods RNA was extracted from dormant M.tuberculosis H37Rv（confirmed by fluorescent dye） and active M.tuberculosis H37Rv.DNA was treated with DNase I and RNA was recovered.mRNA was purified with an mRNA purification kit and then hybridized using a suppression subtractive hybridization（SSH） technique.Differentially expressed genes between dormant M.tuberculosis H37Rv and active M.tuberculosis H37Rv were identified by PCR,cloned,and sequenced.Differentially expressed genes were identified by real-time quantitative PCR.Results Genes that were highly or specifically expressed as testers were obtained with SSH via a forward reaction（dormant M.tuberculosis as tester） and reverse reaction（active M.tuberculosis as tester）.These genes were cloned,sequencing,and subjected to BioEdit and Blast analysis.Fourteen and 17 highly or specifically expressed genes were finally identified in active and dormant M.tuberculosis,respectively.Conclusion Differentially expressed genes between dormant M.tuberculosis H37Rv and active M.tuberculosis H37Rv were identified using an SSH technique and the results have paved the way to the key genes and mechanism of dormancy of M.tuberculosis.