【作者】 乐军；

【导师】 王洪海；

【作者基本信息】 复旦大学 ， 微生物学， 2003， 博士

【摘要】 结核病仍然是威胁人类健康的主要杀手。耐药结核分枝杆菌数量的日益增加和结核合并HIV感染是造成近年来结核病死灰复燃，卷土重来的主要原因。我国不仅是全球结核病的第二大重灾区，也是WHO确定耐多药结核病的高发病率地区。研究结核分枝杆菌耐药机制对于抗结核新药的开发和耐多药结核诊断试剂研制具有重大的意义。为了解中国耐多药结核杆菌的利福平耐药基因rpoB突变特征，我们应用DNA测序技术分析86株结核分枝杆菌临床分离株ropB基因利福平抗药性决定区域(rifampin resistance determining region,RRDR)和V176F突变特征, 72个耐药分离株中的65个菌株中的rpoB基因的RRDR内发现22种不同类型突变, 21种点突变和一个插入突变。最常见突变位点531（41%），526（40%），和 516（4%）。10%耐药分离株未发现突变。新发现RRDR内六个新的等位基因，以及RRDR外部五个新的突变。所有分离菌株均不包含V176突变。其结果有助于发展耐多药结核病快速分子诊断方法和确定我国的耐多药结核病高发热点地区，从而采取有效结核病控制策略。DNA微阵列代表聚合酶链反应产物诊断测序发展方向。由于高密度微阵列价格昂贵，分析操作复杂，和结果难于解释、限制其临床实验室里广泛地运用。我们试图建立一种中等的密度阵列方法，可快速地鉴定结核分枝杆菌利福平耐药菌株。这种方法根据rpoB基因利福平抗药性决定区域内点突变及其他重排的检测。利福平抗药性通过使荧光标记扩增遗传物质与微阵列杂交测定。研究检测53株利福平耐药结核分支杆菌和15株利福平敏感结核分枝杆菌。结果与药物敏感性试验和DNA测序结果一致。PCR扩增仅仅1.5小时可检出利福平耐药临床分离株。寡核苷酸微微阵列是高效方法，可作为检测利福平抗药性的快速方法以弥<WP=5>补标准培养物方法不足。我们首次应用高效液相色谱结合MALDI-MS质谱分析INH耐药和敏感菌株的分泌蛋白的差异，通过DEAE亲和色谱去除培养基上清液牛血清白蛋白后，从异烟肼耐药结核杆菌培养上清液中鉴定一种特征性分泌蛋白，海藻糖磷酸磷酸酶( Trehalose-phosphate Phosphatase，TPP)，分子量46KDa, PI5.3。这是首次报道从结核分枝杆菌的分泌蛋白发现存在TPP。由于海藻糖是分枝杆菌细胞壁的结构完整性所必需的分子，涉及海藻糖糖脂生物合成酶TPP代表潜在抗结核药物靶位。在一般生物体内，TPP一般存在细胞胞浆中，而且TPP与异烟肼耐药相关。从而为血清学水平上快速诊断异烟肼耐药结核病提供一种新的途径。为了鉴定异烟肼耐药结核分枝杆菌特异的蛋白，我们应用双向电泳和MALDI-MS质谱蛋白质组学技术比较分析2株异烟肼耐药结核分枝杆菌和2株敏感结核分枝杆菌。发现异烟肼耐药结核分枝杆菌有26个特征斑点，对其中6个蛋白斑点进行鉴定，6个蛋白分别是海藻糖磷酸磷酸酶（putative trehalose-6-phosphate phosphatase）, 铁钼蛋白moaA, shikimate 5-脱氢酶(putative shikimate 5-dehydrogenase), 葡萄糖胺果糖-6-磷酸氨基转移酶(glucosamine-fructose-6-phosphate aminotransferase), 吲哚-3-甘油磷酸合成酶(indole-3-glycerol phosphate synthase), TetR家族转录调节因子(putative TetR-family transcriptional regulator)。这些酶及调控因子多数为放线菌科关键性的代谢酶和调控因子。研究结果为进一步探索抗分枝杆菌药物和耐药结核病的快速诊断试剂提供线索。 应用高密度cDNA微阵列研究结核杆菌异烟肼耐药菌株与H37Rv异烟肼敏感菌株感染巨噬细胞后差异表达的基因。两者诱导巨噬细胞的差异表达基因有53条，异烟肼耐药菌株比敏感菌株菌株诱导多种趋化因子和细胞免疫增强的细胞因子表达上调和免疫抑制细胞因子IL-10等表达下调。说明异烟肼耐药对细菌毒力有明显的影响，异烟肼耐药菌株的致病力下降，异烟肼耐药菌株容易被宿主免疫系统清除。结果为耐多药结核病控制提供依据。更多还原

【Abstract】 Tuberculosis remains one of the main threats to mankind causing 8 million newcases and 2 million deaths a year. The problem is becoming more critical with the emergence and spread of multidrug resistant(MDR) strains of M.tuberculosis, defined as resistant to at least isoniazid(INH) and rifampin(RIF). China is not only one of the 22 high-burden countries (HBCs) that collectively account for approximately 80% of the world’s tuberculosis cases, but also the hotspot area of very high prevalence of MDR TB identified by WHO(4,5). The increasing prevalence of tuberculosis makes this disease a major focus for drug research and for the development of novel diagnostic tools. Mutations in the 81-bp rifampin resistance determining region(RRDR) and mutation V176F locating in the beginning of the ropB gene were analyzed by DNA sequencing of 86 Mycobacterium tuberculosis clinical isolates(72 resistant and 14 sensitive) from different parts of China. Sixty-five mutations of 22 distinct kinds, 21 point mutations and one insertion , were found in 65 of 72 resistant isolates. The most frequent mutations were in codon 531(41%), 526(40%), and 516(4%). Mutations were not found in seven(10%) of the resistant isolates. Six new alleles within the RRDR, along with five novel mutations outside the RRDR, are reported. None of isolates contained the V176 mutation. The information obtained from this study will assist not only in design of a suitable diagnostic method for rapid detection of MDR TB but also in the identification of any hot-spot regions in the country for proper implementation of TB control strategy. Based on information about ropB gene mentioned above. A moderate density<WP=7>array method that rapidly identified Mycobacterium tuberculosis rifampin resistant strains was developed. The method is based on detection of point mutations and other rearrangements in the rpoB gene region determining rifampin resistance. Rifampin resistance was determined by hybridizing fluorescently labeled, amplified genetic material generated from bacterial colonies to the array. Fifty-three rifampin-resistant M.tuberculosis and fifteen rifampin-sensitive M.tuberculosis were tested, results were concordant with those based on culture drug susceptibility testing and sequencing. Rifampin resistant clinical isolates were detected in as little as 1.5 hour after PCR amplication with visual results. It is demonstrated that oligonucleotide microchip is efficient, specialized technique to implement and could be used as a rapid method for detecting rifampin resistance to complement standard culture-based method.A secreted protein from culture supernatants of INH-resistant M.tuberculosis were purified by HPLC, and identified by MALDI-MS. The protein was trehalose-phosphate phosphatase involved in the biosynthesis of trehalose. Because trehalose is neither synthesized by, nor utilized in, mammalian cells, but is probably an essential molecule for the structural integrity of the mycobacterial cell wall, trehalose-phosphate phosphatase represent excellent potential target sites for chemotherapy against Mycobacterium tuberculosis.We used a proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry to compare the cellular protein composition of two INH resistant strains of Mycobacterium tuberculosis with two INH sensitive strains of Mycobacterium tuberculosis, in order to identify unique proteins of these strains. Emphasis was given to the identification of INH resistant strains specific proteins, because we consider these proteins to represent putative candidates for development of drug and diagnosis of tuberculosis.26 spots were exclusively detected in the INH resistant M.tuberculosis. Six spots of those were identified by MALDI-MS. The results in this study provide a basis for further develop novel anti-mycobacterial agents and diagnostic approach of drug-resistant tuberculosis. The differentially expressed genes in macrophages in response to infection with INH-resistant M.tuberculosis and INH-更多还原