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目的 研究国内临床耐氟康唑白念珠菌的吡咯类药物作用靶酶———细胞色素P 45 0羊毛固醇 14 α脱甲基酶的基因ERG11突变。方法 用酶消化和碱裂解法抽提基因组DNA ,PCR扩增ERG11基因的读码框片段 ,将目的片段与PBS载体 (BluscriptM 13)连接 ,重组体转入DH5α中 ,从而测定序列。结果 在 15株耐药菌中共发现了 12个有义点突变、17个无义点突变和一个菌株的部分移码。 12个有义点突变的氨基酸变异是F72L、D81G、D116E、K12 8T、Y132H、E2 6 6D、D2 94G、S36 1P、M 374V、P386L、H40 0R和Q474K ,突变主要靠近羧基端 ;而敏感株的氨基酸变异为F72L、K12 8T和E2 6 6D ,靠近氨基端。耐药株的D2 94G、S36 1P、M374V、P386L、H40 0R和Q474K氨基酸变异与文献报道不同。结论 耐药株和敏感株的点突变不同 ,D2 94G、S36 1P、M374V、P386L、H40 0R和Q474K变异位点可能与耐药性有关 ,做整段编码基因的功能表达 ,有助于明确变异与耐药的关系
Objective To investigate the ERG11 gene mutation of the target gene of pyrrole-resistant drug-resistant Candida albicans to clinical use of cytochrome P 45 0 lanosterol 14 α demethylase. Methods Genomic DNA was extracted by enzymatic digestion and alkaline lysis. The reading frame of ERG11 gene was amplified by PCR. The target fragment was ligated into PBS vector (Bluscript M 13) and the recombinant was transferred into DH5α to determine the sequence. Results A total of 12 sense point mutations, 17 non-sense point mutations and partial frameshift of one strain were found in 15 resistant strains. The amino acid variations of 12 sense point mutations were F72L, D81G, D116E, K12 8T, Y132H, E2 6 6D, D2 94G, S36 1P, M 374V, P386L, H40 0R and Q474K, The amino acid variations were F72L, K12 8T and E2 6 6D, near the amino terminus. D294G, S36 1P, M374V, P386L, H40 0R and Q474K amino acid variations of resistant strains were different from those reported in the literature. Conclusion The point mutations of drug-resistant and susceptible strains are different. The D294G, S36 1P, M374V, P386L, H40 0R and Q474K mutation sites may be related to drug resistance, so the functional expression of the entire coding gene contributes to the definite mutation Relationship with resistance