简并PCR结合RACE法克隆希木龙念珠菌CDR1基因  

Cloning the Drug-efflux-pump-encoding genes( CDR1) from Two Different Candida Haemulonii Strains with Degenerate PCR Combined RACE

作　　者：张浩[1] 王千[1] 周亚彬 李若瑜[1] 刘伟[1] 

机构地区：[1]北京大学第一医院皮肤性病科北京大学真菌和真菌病研究中心皮肤病分子诊断北京市重点实验室,北京100034

出　　处：《中国皮肤性病学杂志》2018年第1期29-32,共4页The Chinese Journal of Dermatovenereology

基　　金：国家自然科学基金(81471925)

摘　　要：目的克隆希木龙念珠菌中药外排泵基因(CDR1),为探讨希木龙念珠菌的耐药机制奠定基础。方法首先从NCBI基因数据库中找到已知的白念珠菌、热带念珠菌、近平滑念珠菌和光滑念珠菌Cdr1蛋白的氨基酸保守序列,并设计简并引物,PCR扩增获得希木龙念珠菌CDR1c DNA部分片段,接着使用RACE方法分别扩增其5'和3'端序列得到完整的CDR1编码序列(CDS)。结果简并PCR扩增得到预期2 210bp大小的片段;5'RACE和3'RACE分别得到CDR1c DNA 5'和3'端片段,经纯化、克隆、测序、比对和拼接分别得到两株菌完整的CDR1c DNA序列;其编码的蛋白序列经比对发现与其它念珠菌的Cdr1蛋白高度同源。结论利用简并PCR联合RACE方法能够有效、准确地克隆出希木龙念珠菌CDR1基因。Objective CDR1genes from two differentCandida haemulonii strains were cloned for studying the mechanism of antifungal resistance.Methods To obtain CDR1 gene,degenerate primers were designed according to the conserved sequences in Cdr1 protein from the other four types of Candida spp.Partial CDR1cDNA were amplified by degenerate PCR.And 5＇ cDNA and 3＇cDNA fragments were amplified by RACE method.The full-length CDR1 coding sequence（CDS）were obtained after aligning and splicing.Results The results showed the complete CDR1 CDS were obtained and identified through homologous alignment of Cdr1 proteins with the other Candida.Conclusion This study suggested CDR1genes can be effectively cloned by degenerate PCR combined RACE.

关 键 词：希木龙念珠菌 简并PCR 快速cDNA末端扩增法 药物外排泵 

分 类 号：R756[医药卫生—皮肤病学与性病学] R372[医药卫生—临床医学]