样品制备<em>结核分枝杆菌</em提取物对核磁共振的代谢组学研究
Summary
代谢组学的档案<em>结核分枝杆菌</em>确定后在肉汤培养物的生长。条件可以改变，以测试的营养补充剂，氧化剂，抗结核剂的影响，对这种微生物的代谢的档案。提取物制剂的程序是适用于两个1D<sup> 1</sup> H和2D<sup> 1</sup> H-<sup> 13</sup> C NMR分析。
Abstract
结核分枝杆菌是在全球范围内人类死亡的一个重要原因。两个多（MDR）和广泛耐药（XDR）株的出现，威胁要破坏当前疾病控制工作。因此，迫切需要开发比现有的，更有效的药物和疫苗。 分枝杆菌的基因组肺结核已超过10年，但在我们的基因的功能和重要性的认识有重要的差距。许多研究都因为基因表达的转录组和蛋白质组水平的分析，以确定影响的药物，氧化剂，对全球基因表达模式和生长条件。最终，这些变化的最终响应反映在代谢的细菌组合物，包括几千小分子量的化学品。比较野生型和突变株的代谢概况，无论是未经处理或者tr与一个特定的药物eated，可以有效地允许的目标识别和可能导致具有反结核活性的新型抑制剂的发展。同样地，对代谢组中的两个或多个条件的影响也可以被评估。核磁共振（NMR）是一个功能强大的技术，用于识别和量化的中间代谢产物。在这个协议中，程序编制M.结核病细胞提取物的NMR代谢组学分析进行了阐述。在适当条件下，所需的生物安全级别3遏制，1收获细胞培养物生长，并进行机械裂解，同时保持冷的温度，以最大限度地提高保存的代谢物。细胞裂解物中回收，过滤灭菌，并存储在超低温度下。从这些细胞提取物的等分试样镀米德尔7H9琼脂集落形成单位验证没有存活的细胞上。后两个月的孵育在37℃下，如果没有㈥能够观察到菌落，取出样品，从包容设施用于下游处理。萃取液冷冻干燥，再悬浮在氘化缓冲液中喷射的NMR仪器，捕获分光数据，然后将其进行统计分析。所描述的方法可以应用于两个一维（1D）的1 H NMR和二维（2D）的1 H-13 C-NMR分析。这种方法提供了更可靠的小分子量代谢物鉴定和色谱法比更可靠，更敏感的细胞提取液代谢成分的定量分析。描述的过程中的变化之后的细胞裂解步骤也可以适于并行蛋白质组学分析。
Protocol
1。协议文本该协议强调的NMR方法，以适应M.肺结核 （III级代理）。因此，必须遵循生物安全3级（BSL3）的做法时，M.肺结核在每年认证的实验室研究。暴露于实验室产生的气溶胶是最重要的工作人员与这些微生物所遇到的危险。下面的过程是在我们的机构中进行的变化可能存在生物安全委员会的建议的基础上。常见的个人防护设备包括特卫强服，圆?…
Discussion
一个相当多的研究分析，转录组和蛋白质组的配置文件的M.在体外和体内条件下各种肺结核 11-16最终，在基因表达和酶活性的变化导致在分子量小的分子的浓度的变化。的完整描述，这些化合物构成的代谢组。因此，药物和不同生长条件对代谢途径的影响可能其次是代谢组学分析。17,18很少有研究利用这种方法，研究代谢途径中的M.结核 （见下文）和其?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者想感谢所有成员的博士巴列塔，博士的实验室权力的有益的意见而开发的协议。我们温迪奥斯汀的有益讨论和校对的稿子。这个手稿中描述的工作是由种子试点补助金每个上面列出大学内布拉斯加 – 林肯氧化还原生物学研究中心（母公司授予＃的NCRR 2P20RR 017675，D.贝克尔，PI）的研究者。此外，我们感谢奥费利娅查孔博士的研究用品和先生Halouska的部分工资支持，以规范本出版物中包含的NMR技术从她的R21授予（1R21AI087561-01A1）提供资金。
Materials
| Name of the Reagent/Equipment | Company | Catalogue Number | Comments |
| ADC Enrichment | BD BBL Middlebrook | 212352 | |
| BACS-120 Sample Changer | Bruker | ||
| Bruker Avance NMR | Bruker | 500 MHz | |
| Bovine Serum Albumin | Fisher Scientific | BP1600-100 | Fraction V |
| Centrifuge | Beckman Coulter | Allegra X-15R | Benchtop |
| Centrifuge Tubes | Corning | 430291 | 50 ml sterile polypropylene |
| Cryogenic Vials | Corning | 430488 | 2.0 ml sterile polypropylene |
| Cycloheximide | A.G. Scientific | C-1189 | Toxic |
| D(+) – Glucose | ACROS | 41095-0010 | |
| Deuterium Oxide | Sigma Aldrich | 617385 | |
| Erlenmeyer Flask | VWR | 89095-266 | Sterile, flat base, polycarbonate, 0.22 μm PTFE membrane vented cap |
| Flash Freeze Flask | VWR | 82018-226 | 750 ml |
| Freeze Dryer | VWR | 82019-038 | 4.5 L Benchtop |
| Glycerol | GibcoBRL | 15514-029 | |
| Incubator | New Brunswick | Innova 40 | Benchtop shaker |
| Lysing Matrix B | MP Biomedicals | 6911-100 | |
| Lysis Machine | MP Biomedicals | FastPrep-24 | |
| Microcentrifuge | Eppendorf | 5415D | Benchtop |
| Microcentrifuge | Beckman Coulter | Microfuge 22R | Benchtop |
| Middlebrook 7H9 Broth | Difco | 271310 | |
| NMR tubes | Norell | ST500-7 | 5mM |
| OADC Enrichment | BD BBL Middlebrook | 212351 | |
| Oleic Acid | Sigma | O1008 | |
| Potassium Phosphate Dibasic | VWR | BDH0266 | |
| Potassium Phosphate Monobasic | VWR | BDH0268 | |
| Rotor – Microfuge 22R | Beckman Coulter | F241.5P | Sealed and polypropylene |
| Rotor – Allegra X-15R | Beckman Coulter | SX4750 | With bio-certified covers |
| Sodium Chloride | Fisher Scientific | S271-3 | |
| Sodium-3-trimethylsilylpropionate-2,2,3,3-D4 | Cambridge Isotope | DLM-48 | |
| Spectrophotometer | Beckman Coulter | DU-530 | |
| Spectrophotometer Cuvettes | LifeLINE | LS-2410 | 1.5 ml polystyrene, 2 clear sides |
| Syringe | Becton Dickinson | 309585 | Sterile, 3 ml Luer-Lok |
| Syringe Filter | Nalgene | 190-2520 | 0.2 μm sterile cellulose acetate |
| Tween 80 | Fisher Scientific | BP338-500 |
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Citer Cet Article
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