氨基糖苷-(3)-乙酰转移酶II的表达纯化及初步活性  

Expression and Purification of AAC(3)-II,a Aminoglycoside Acetyltransferase,and Its Activity

作　　者：王震[1] 陈云波[1] 查长森[1] 邹立林[1] 彭颖[1] 季敬璋[1] 陈秀枢[2] 吕建新[1] 

机构地区：[1]温州医学院浙江省医学遗传学重点实验室 [2]温州医学院微生态学研究所,温州325035

出　　处：《细胞生物学杂志》2007年第1期81-85,共5页Chinese Journal of Cell Biology

基　　金：浙江省科技厅重点项目(No.2004C23018)~~

摘　　要：用PCR扩增的氨基糖苷-(3)-乙酰转移酶II[aac(3)-II]基因构建了能表达较高活性AAC(3)-II的重组工程菌,并获取重组AAC(3)-II。含pET28a质粒的工程菌转入aac(3)-II基因前后对氨基糖苷类抗生素的最低抑菌浓度进行比较,重组菌超声裂解上清液及其纯化的AAC(3)-II进行SDS-PAGE和Western印迹电泳鉴定。最低抑菌浓度表明转入aac(3)-II基因的工程菌比未转入的工程菌对庆大霉素(gentamicin,GEN)的提高了256倍,对妥布霉素(tobramycin,TOB)及其奈替米星(netilmicin,NTL)提高了16倍,电泳鉴定表明纯化获取的是重组AAC(3)-II,其相对分子质量约为32kDa,纯度大于95%,纯化后的10μgAAC(3)-II,分别在10s内使80μgGEN、30s内使80μgTOB和NTL乙酰化而失去抑菌作用。研究为氨基糖苷类抗生素伴侣的开发研究初步打下基础。The aac（3）-Ⅱ was amplied by PCR and cloned into pET28a expression plasmid, then the recombinant plasmid was transformed into E.coli BL21（DE3）, lastly AAC（3）-Ⅱ was purified from the recombinant bacteria. The activity of AAC（3）-Ⅱ was analyzed by comparing minimum inhibitory concentration （MIC） between the recombinant bacteria owning aac（3）-Ⅱ and not. Bacteria were disrupted by ultrasonic treatment. Bacteria debris was removed by centrifugation, and the supematant and AAC（3）-Ⅱ purified from the supematant were analyzed by SDS-PAGE and Western Blot electrophoresis. Results demonstrated that the bacteria owning aac（3）-Ⅱ were 256-fold higher in gentamicin （GEN） MIC, 16-fold higher in tobramycin （TOB） and netilmicin （NTL） MIC than the bacteria not owing aac（3）-Ⅱ. SDS-PAGE and Western blot electrophoresis proved purified protein was AAC（3）-II that its molecular weight was 32.0 kDa, purity of that was more than 95 percentage. The purified 10 lag AAC（3）-Ⅱ transfered acetyl to GEN in 10 s, to TOB and NTL in 30 s, so the antibiotics were invalidated. The research provided fundamental conditions for exploiting the drug eliminating aminoglycosides.

关 键 词：氨基糖苷乙酰转移酶 最低抑菌浓度 纯化 氨基糖苷类抗生素 

分 类 号：Q78[生物学—分子生物学]