细胞壁缺陷结核分枝杆菌耐药性基因探究.docVIP

细胞壁缺陷结核分枝杆菌耐药性基因探究 　 作者：王和 罗振华 徐艳 梁菁苹 【摘要】 目的 检测与分析细胞壁缺陷结核分枝杆菌的耐药性相关基因，探讨细胞壁缺陷结核分枝杆菌耐药性的基因基础。方法 分别分离16株自发形成和用利福平、异烟肼、乙胺丁醇诱导形成的结核分枝杆菌的染色体DNA，PCR扩增IS986序列以及rpoB、katG和embB基因后进行凝胶电泳和序列分析。结果 自发形成及抗结核药物诱导形成的结核分枝杆菌稳定L型可在含利福平(4μg/ml)、异烟肼(0.2μg/ml)、乙胺丁醇(7.5μg/ml)的培养基内生长与传代培养。基因检测与分析显示，结核分枝杆菌稳定L型仍然保留IS986基因，rpoB、katG、embB基因也未见突变。结论 结核分枝杆菌稳定L型可自发形成，也可被利福平、异烟肼和乙胺丁醇诱导形成。结核分枝杆菌稳定L型对利福平、异烟肼、乙胺丁醇形成耐药性，但其染色体上耐药相关基因并没有发生突变。除染色体耐药相关基因突变可造成结核分枝杆菌形成耐药性外，细胞壁缺陷也是造成结核分枝杆菌形成耐药性的一个重要机制。 【关键词】 结核分枝杆菌； 细胞壁； 基因； 耐药性 ABSTRACT Objective To determine and analyze the genes related to drug resistance, and probe the properties of drug resistance genes of Mycobacterium tuberculosis. Methods The sequence of IS986 and the genes of rpoB, katG and embB were isolated respectively from the L-forms derived spontaneously and induced by rifampin, isoniazid and ethambutol respectively from 16 strains of M.tuberculosis, and then the genes were determined by agarose gel electrophoresis and analyzed by direct sequencing. Results The both of L-forms derived spontaneously and induced from M.tuberculosis were grow well and be subcultured in the non-high osmotic liquid media with rifampin (4μg/ml), isoniazid (0.2μg/ml) and ethambutol (7.5μg/ml) respectively. The sequence of IS986 and the genes of rpoB, katG and embB were held in the stable L-forms and no gene mutation was found in them. Conclusion The L-forms were formed spontaneously or induced by rifampin, isoniazid and ethambutol. The L-forms derived either spontaneously or induced by the antibacterial drugs from drug susceptible strains of M.tuberculosis revealed the resistance to rifampin, isoniazid and ethambutol but no gene mutation of rpoB, katG and embB could be found in the variants. It was considered that the cell wall deficiency was an important resistant mechanism of M.tuberculosis besides the chromosomal gene mutation. KEY WORDS Mycobacterium tuberculosis; Cell wall; Gene; Drug resistance 细胞壁缺陷是结核分枝杆菌最常发生的变异现象之一，被认为是结核分枝杆菌生命周期独特的一个形态时期或相［1］。然而近年的研究［2