耐伊曲康唑克柔念珠菌ERG11基因突变分析

发布时间：2019-06-27 15:12

【摘要】：随着广谱抗生素、免疫抑制剂的广泛应用和介入疗法、器官移植等的深入开展,侵袭性真菌感染的发病率不断增高,尽管白念珠菌仍是临床最为常见的真菌,但非白念珠菌的分离率不断升高,而抗真菌药物的广泛应用则加速了菌株的耐药进程,非白念珠菌临床分离株中耐药株的比例明显增加,非白念珠菌耐药问题逐渐引起人们的重视。从分子水平研究非白念珠菌对抗真菌药尤其是唑类药物的耐药机制是目前的热点。 ERG11是羊毛甾醇14a-去甲基化酶的编码基因,14a-去甲基化酶是念珠菌细胞膜麦角固醇合成途径中的关键酶,对维持细胞膜的正常结构和功能具有重要意义。唑类抗真菌药物分子可与14a-去甲基化酶分子结合,阻断羊毛固醇去甲基化过程,影响麦角固醇合成。ERG11的突变可以改变14a-去甲基化酶的氨基酸序列,当这种改变足以影响酶分子的空间构型时,酶分子与药物分子间的亲和力可能被削弱,导致菌株耐药。目前的研究主要针对白念珠菌,对于克柔念珠菌国内外均未见相关文献报道。目的：研究克柔念珠菌临床分离菌株麦角固醇合成途径中关键酶编码基因ERG11突变与耐唑类药物伊曲康唑之间的关系。方法：选择2009年6月至2010年10月从本院临床科室送检标本中分离得到的来自痰液、尿液、阴道分泌物的6株克柔念珠菌伊曲康唑耐药株和3株敏感株,1株ATCC6258克柔念珠菌标准株(购自北大一院真菌中心),以抽提的克柔念珠菌基因组DNA为模板,对ERG11基因序列进行PCR扩增,PCR产物直接DNA序列测定,最后应用BLAST软件对测序结果进行比较。结果：10株克柔念珠菌的ERG11基因序列(以已知序列第1个ATG起始密码的A作为第1个记数单位)共有7个位点发生突变,均为同义突变位点。在第447bp位点耐药菌株和敏感株均发生碱基突变。在第12bp、327bp、429bp、552bp位点耐药菌株发生碱基突变,在第696bp、927bp位点敏感菌株发生碱基突变。结论：本研究证实克柔念珠菌ERG11基因序列存在多态性,未发现因ERG11基因突变引起靶酶氨基酸改变而导致的克柔念珠菌对伊曲康唑耐药。
[Abstract]:With the wide application of broad-spectrum antibiotics, immunosuppressants, interventional therapy, organ transplantation and so on, the incidence of invasive fungal infection is increasing. Although candida albicans is still the most common fungus in clinic, the isolation rate of non-candida albicans is increasing, while the wide application of antifungal drugs accelerates the process of drug resistance, and the proportion of drug-resistant strains in non-candida albicans clinical isolates is significantly increased. The drug resistance of non-candida albicans has been paid more and more attention. It is a hot spot to study the resistance mechanism of non-candida albicans antifungal drugs, especially Azoles, at the molecular level. ERG11 is the coding gene of lanosterol 14a-demethylase. 14a-demethylase is the key enzyme in the synthesis of ergosterol in candida cell membrane, which is of great significance to maintain the normal structure and function of cell membrane. Zole antifungal molecules can bind to 14a demethylase molecules, block the demethylation process of wool sterol and affect ergosterol synthesis. ERG11 mutation can change the amino acid sequence of 14a demethylase. When this change is enough to affect the spatial configuration of enzyme molecules, the affinity between enzyme molecules and drug molecules may be weakened, resulting in drug resistance. At present, the research is mainly aimed at candida albicans, but there is no related literature report on candida albicans at home and abroad. Aim: to study the relationship between the ERG11 mutation of the key enzyme coding gene in ergosterol synthesis pathway and itraconazole. Methods: from June 2009 to October 2010, 6 strains of itraconazole resistant strains and 3 sensitive strains of C. roxburghii isolated from sputum, urine and vaginal secretions, and one standard strain of ATCC6258 (purchased from the fungal center of the first hospital of Peking University) were selected. Using the extracted genomic DNA of candida cerevisiae as template, the ERG11 gene sequence was amplified by PCR and the PCR product was sequenced directly. Finally, BLAST software is used to compare the sequencing results. Results: a total of 7 loci were mutated in the ERG11 gene sequence of 10 strains of candida cerevisiae (using A of the first ATG initiation code of the known sequence as the first counting unit), all of which were synonymous with each other. Base mutations occurred in both drug-resistant and sensitive strains at 447 BP site. At 12bp, 327bp, 429bp, 552 BP resistant strain and 696bp, 927bp site sensitive strain had base mutation. Conclusion: this study confirmed that there was polymorphism in the ERG11 gene sequence of candida krooensis, and no resistance to itraconazole was found due to the change of amino acid in the target enzyme caused by the mutation of ERG11 gene.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R379

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