依赖利福平结核分枝杆菌相关蛋白的氨基酸及其编码基因序列研究  

Study on the sequences of amino-acids and their gene codes of the related proteins of rifampin-dependent Mycobacterium tuberculosis

作　　者：钟敏[1] 王易伟[1] 胡频频[1] 包佳玲[2] 钟静[1] 胡忠义[3] 罗永艾[4] 

机构地区：[1]重庆市肺科医院重庆市结核基因诊断中心实验室,400020 [2]复旦大学生命科学院 [3]上海市肺科医院 [4]重庆医科大学第一临床学院肺科

出　　处：《中华结核和呼吸杂志》2006年第2期83-86,共4页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：2003重庆市科委医学应用基础研究项目(8014);重庆市卫生局医学重点项目(031010)

摘　　要：目的研究临床依赖利福平结核分枝杆菌(依R菌)相关蛋白的氨基酸及其编码基因序列。方法以罗氏绝对浓度法药敏实验鉴定临床依R菌株(49株)、耐R菌株(54株)和R敏感菌株(30株),用十二烷基硫酸钠聚丙烯酰胺电泳(SDSPAGE)法对不同临床菌株及H37Rv标准株共计134株菌株的菌体蛋白质图谱进行比较分析,通过QTOF2型毛细管液相色谱电喷雾四极杆飞行时间串联质谱仪(LCESIMSMS)对其差异表达蛋白的氨基酸序列进行鉴定,并用对其蛋白的编码基因进行DNA序列分析。结果不同菌株的SDSPAGE菌体蛋白图谱基本相似。49株依R菌有41株(84%)在含R管中的菌体蛋白图谱有一条相同的高表达蛋白带(相对于标准物分子质量43000的下端),约占总菌体蛋白的30%～50%,H37Rv标准株、28株R敏感菌(28/30)及44株耐R菌(44/54)的含R管和对照管的蛋白图谱相似,且无相应的高表达现象;该高表达蛋白经氨基酸序列鉴定为结核分枝杆菌H37Rv假设蛋白Rv0341,相对分子质量为43894,等电点(PI)5.25,得分183。不同菌株的Rv0341编码基因1440bp扩增片段,经DNA测序分析均未发现突变。结论利福平对Rv0341在依R菌中的表达有上调作用,Rv0341与依R菌相关,可视为依R菌的相关蛋白或标志物;Rv0341与依R菌的毒力及细胞壁的合成是否相关有进一步的研究价值。Objective To study the sequences of amino-acids and their related genes of rifampin dependent Mycobacterium tuberculosis. Methods The strains of rifampin-dependent, rifampin-resistant and rifampin-sensitive Mycobacterium tuberculosis were evaluated by L-J method. The proteins of 134 strains were analyzed by sodium dodecyl sulfate （SDS）-polyacrylamide gel electrophoresis （SDS-PAGE）. The sequences of amino-acids of the proteins with differential expressions were determined by Q-TOF2 liquid chromatography-electra mist spray- quadrupole -time of flight cascade mass spectrograph （ LC-ESI-MS-MS, Q-TOF2）. The corresponding gene codes for the proteins were analyzed. Results The protein SDS-PAGE profile of 41 of 49 （41/49） rifampin-dependent strains showed a high expression of a protein band （molecular weight 43 000）, representive of 30% -50% of the total proteins differed from rifampin restance and the control strains. The protein profile of H37Rv standard strains, 28 （28/30） rifampin-sensitive strains and 44 （44/54） rifampin-resistant strains showed no relevant high expression. The protein of high expression was identified as the hypothetical protein Rv0341 of Mycobacterium tuberculosis H37Rv with a molecular weight （M） of 43 894, a iso-potential point of 5.25 and a score of 183. No mutation was found in the 1 440 bp encoding sequence in various strains. Conclusions The hypothetical protein Rv0341 is related to the rifampin-dependent strains, so it can be regarded as an indicator for rifampin-dependent Mycobacterium tuberculosis. Rifampin upregulated the expression of Rv0341 in rifampin-dependent Mycobacterium tuberculosis. Whether the hypothetical protein Rv0341 is related to virulence and synthesis of the cell wall of Mycobacterium tuberculosis needs further study.

关 键 词：分枝杆菌 结核 利福平 蛋白质组 基因 

分 类 号：R378[医药卫生—病原生物学]