结核分枝杆菌休眠复苏期与活跃期的差异表达基因分析  

Analysis of differentially expressed genes between resuscitating and active Mycobacterium tuberculosis

作　　者：刘忠泉[1] 张宗德[1] 邢爱英[1] 陈曦[1] 李自慧[1] 古淑香[1] 贾红艳[1] 杜博平 马玙[1] 

机构地区：[1]北京市结核病胸部肿瘤研究所结核病分子生物学研究室,101149

出　　处：《中华结核和呼吸杂志》2008年第6期442-447,共6页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：北京市自然科学基金资助项目（7052014）

摘　　要：目的寻找休眠结核分枝杆菌复苏的关键基因，探讨结核分枝杆菌复苏的机制。方法取改良7H9培养基培养20d的结核分枝杆菌标准株H37Rv作为对数生长期的活跃结核分枝杆菌，提取其RNA。将一部分该菌株在37℃密封无氧培养45d左右，加入亚甲蓝作为无氧标志，即获得休眠菌。向休眠菌中加入复苏促进因子，37℃有氧培养3d，提取休眠复苏期结核分枝杆菌的基因组RNA。用DNA酶处理RNA并回收RNA，用mRNA纯化试剂盒纯化RNA。利用抑制性消减杂交（Suppression subtractive hybridization，SSH）技术分析休眠复苏期和活跃期结核分枝杆菌的基因组mRNA的表达差异，并通过基因克隆、测序和序列分析，寻找差异表达基因。实时荧光定量PCR鉴定差异表达基因。结果以活跃期结核分枝杆菌cDNA为检测子的正相杂交和以休眠复苏期结核分枝杆菌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了选择性扩增，杂交产物经连接pTA2载体、转化至大肠杆菌DH5α和蓝白斑筛选，正相杂交获得78个阳性菌落，反相杂交获得46个阳性菌落。阳性单个菌落经液体LB培养基培养，以菌液为模板，T7、M13为引物，扩增插入片段，能扩增到明显的条带，且〉350bp的条带为阳性。正相杂交得到66个阳性扩增带，反相杂交得到39个阳性扩增带。这105个阳性扩增带的菌液经测序分析，则正相有30个特异性序列，反相有21个特异性序列。将这51个序列通过Genbank检索，因有不同序列对应同一基因，最后正相获得了20个活跃结核分枝杆菌特异性表达或高表达的功能基因，反相获得了7个休眠复苏期结核分枝杆菌特异性表达或高表达的功能基因，根据其功能不同分为8大类。经实时荧光定量PCR分析，7个休眠复苏期结核分枝杆菌特异性表达或高表达功能基因的表达量均为活跃结核分枝杆菌的该基因表达�Objective To screen key genes of dormant M. tuberculosis for resuscitation. Methods M. tuberculosis H37Rv strain cultured for 20 days in 7H9 liquid medium was used as active bacteria. Dormant bacteria were obtained by cultivating active bacteria hermetically at 37℃ using methylene blue as the indicator of oxygen free until the blue medium became colorless. Then resuscitation promoting factors were added to the culture and the bacteria were cultivated for 3 days to be resuscitated. RNA was extracted from active bacteria and resuscitating bacteria, disposed of DNA in RNA with DNase Ⅰ , and mRNA was purified and then were hybridized using suppression subtractive hybridization （SSH） technique. Differentially expressed genes between resuscitating M. tuberculosis and active M. tuberculosis were identified by PCR, cloning, and sequence alignment. Identification of the differentially expressed genes was performed by real-time quantitative PCR. Results High or specifically expressed genes as tester had been obtained by SSH in correctitude reaction （ active M. tuberculosis as tester） and reverse reaction （ dormant M. tuberculosis as tester）. These genes were cloned into plasmid PGEM-T Easy, and 78 positive bacteria in correctitude reaction and 46 positive bacteria in reverse reaction were obtained. The positive bacteria were amplified by PCR with T7 and M13 primer, and 66 positive bacteria （ 〉350 bp） in correctitude reaction and 39 positive bacteria（ 〉 350 bp ） in reverse reaction were obtained. After sequencing, 30 positive sequences in correctitude reaction and 21 positive sequences in reverse reaction were obtained. Twenty and 7 high or specifically expressed genes were finally identified in active and resuscitating M. tuberculosis respectively by seaiching in Genbank. These genes were classified into 8 categories. Real-time quantitative PCR demonstrated that the quantity of 7 high or specifically expressed genes in resuscitating bacteria was more than 4 times that in active bacteria. Conclus

关 键 词：分枝杆菌 结核 基因 抑制性消减杂交 

分 类 号：R686[医药卫生—骨科学]