唑类耐药白色念珠菌羊毛甾醇14α-去甲基化酶基因的研究

目的探讨白色念珠菌羊毛甾醇14α-去甲基化酶（CYP51）基因突变与对唑类抗真菌药物耐药的关系,从分子水平了解其耐药机制。方法纸片扩散法和NCCL公布的M-27方案测定耐药株对氟康唑和伊曲康唑的MIC;设计引物,PCR扩增唑类耐药白色念珠菌的CYP51基因;扩增产物测序并与Genbank序列相比较分析。结果扩增产物测序分析表明,成功扩增到白色念珠菌CYP51基因。与X13296株序列相比较,两个耐药株都存在有意义突变和无意义突变。两株菌共有22个碱基突变。与以往报道的相同,突变发生氨基酸替换的有F105L、K128T、Y133H、T199I、R267H、G464S和G467K。其中,两株菌都有Y132H和G467K突变。F71L、W244R、T311N和T352I为新发现的突变,未见报道。同时,也发现了9个未发生氨基酸替换的突变。结论白色念珠菌对唑类抗真菌药物的耐药与CYP51基因突变有关,且为多位点突变。

Study on the point mutations of lanosterol14-alpha-demethylase (CYP51) gene of azole-resistant Candida albicans isolates by sequencing analysis

Objective To explore the relationship between the point mutations of CYP51gene and the azole-resistance mechanism in Candida albicans clinical isolates.Methods The paper diffusion test and NCCLS M-27 protocols were used to screen the fluconazole-resistant and itraconazole resistant Candida albicans clinical isolates.CYP51 gene of two azole-resistant C.albicans clinical strains were amplified by three pairs of primers,respectively.The PCR products purified were sequenced,and compared with the nucleotide sequences of C.albicans（accession no：X13296） to find out the mutation sites.Results The nucleotide sequence analysis showed that there were both significan point mutions and silent mutations in CYP51 gene from two azole-resistant iasolates of C.albicans.Seven mutations previously described,F105L,K128T,Y132H,T199I,R267H,G464S,and R467K,were identified in the two strains.The animo acid substitutions of Y132H and R467K,known to contribution to azole resistance,were detected in both 2007H strain and 2007T strain.Four noval mutations,including F71L,W244R,T311N and T352I,were simutinously identified.Nine silent mutations appeared in two isolates,also.Conclusion In this survey,the two azole-resistant C.albicans clinical isolates contained more than one mutations in CYP51 gene that are associated with azole resistance.Four novel mutations of CYP51 may be associated with the resistance of C.albicans to azoles.And the mechanisms need to be further studied in detail.