摘要
利用错配内部引物 ,采用重组PCR方法获得H31 0D突变型白念珠菌 1 4α 去甲基化酶(CYP5 1 ) ,构建H31 0D突变型CYP5 1蛋白的表达载体pYCYP5 1M ,转化进入酵母菌INVSC - 1中 ,半乳糖诱导蛋白的表达 ,微量液基稀释法测定表达野生型及突变型CYP5 1蛋白的宿主酵母菌对氟康唑的MIC。表达的CYP5 1蛋白占微粒体蛋白酶系的 1 5 % ;表达突变蛋白的酵母菌MIC值是表达野生蛋白的酵母菌MIC值的 2倍。CYP5 1蛋白H31 0D的突变导致表达突变蛋白的酵母菌对氟康唑MIC的升高 ,证明H31 0残基对CYP5 1蛋白与氟康唑的结合有一定作用 ,为研究新型抗真菌前导化合物寻找新的靶点。
The method of recombinant PCR was employed to obtain H310D mutation in 14α-demethylase in Candida albicans ). The expression vector pYCYP51M harboning the mutant CYP51M gene was constructed. The mutant CYP51 was expressed in Sacchromyces cerevisiae induced by 2% galactose. At last the MICs of yeasts to Fluconazole were determined by broth microdilution testing. Expressed protein was 15% of the total microsome proteins. The MICs of yeasts expressed the mutant CYP51 were 4-fold as that of the yeasts expressed the wild CYP51. The increase of the MICs was due to the alteration H310D in CYP51. It could be concluded that the residue of H310 was important to the function of CYP51.
出处
《微生物学报》
CAS
CSCD
北大核心
2003年第6期712-716,共5页
Acta Microbiologica Sinica
基金
国家自然科学基金 (3 9770 876)
上海市科技发展基金 (98QB1 40 0 9)~~
