耐氟康唑白念珠菌ERG11的基因突变

The point mutation of cytochrome P-450 lanosterol 14-α demethylase ERG11 gene in Fluconazole-resistant Candida albicans

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 。

Objective To investigate the point mutation of the open reading frame of cytochrome P-450 lanosterol 14-α demethylase gene ERG11. Methods In order to identify such alterations, the DNA was extracted by enzyme lysis methods and the PCR was performed. The PCR products were purified and cloned into PBS vector, then transformed into DH5α and sequenced. Results Twenty nine point mutations were identified in this 15 resistant isolates, most of which were different from susceptible strains. The mutations included 12 missense substitutions: F72L, D81G, D116E, K128T, Y132H, E266D, D294G, S361P, M374V, P386L, H400R, and Q474K, of which 6 had not been described previously (D294G, S361P, M374V, P386L, H400R, and Q474K). The other mutations included a frameshift, and 17 silent mutations. Conclusions The point mutation of resistant isolate is different from that of the susceptible isolate. It is suggested that the drug resistance is related with by the newly found alterations, including D294G, S361P, M374V, P386L, H400R, Q474K, and a frameshift.