摘要
目的构建CTX-M型超广谱β-内酰胺酶基因突变体,比较突变前、后酶活性的变化,进一步阐明酶的分子结构特征和生化特性。方法利用重叠延伸PCR定点诱变技术将CTX-M型超广谱β-内酰胺酶基因Ser240(丝氨酸)AGC定点突变为Gly240(甘氨酸)GGC,分别构建CTX-M型酶的原核重组表达载体及其突变表达载体,重组载体经酶切鉴定和DNA测序鉴定后,转化大肠埃希菌JM109,比较突变前、后酶活性的改变。结果酶切鉴定和DNA测序鉴定表明CTX-M型酶的原核重组表达载体及其突变表达载体构建成功,转化大肠埃希菌JM109后,发现含野生型CTX-M基因的重组菌其酶活性与来源菌株相近,而含突变型CTX-M基因的重组菌未检测到酶活性。结论Ser240对于维持CTX-M型超广谱β-内酰胺酶活性具有重要作用。
Objective To construct the S240G mutant of extended-spectrum β-lactamase CTX-M, and study compare the enzyme activity of CTX-M before and after mutagenesis. Methods The CTX-M gene and the mutant CTX-M gene were obtained using Polymerase Chain Reaction （PCR）. The gene fragments were cloned into plasmid pBK-CMV to construct recombinant plasmid. After identified by DNA sequencing, the β-cloned into plasmid pBK-CMV to construct recombinant plasmid. After identified by DNA sequencing, the lactamases were extracted from E. coli JM109 transformed by recombinant plasmids and the enzyme activities were detected. Results Two recombinant plasmids were successfully constructed. β-lactamase of wild CTX-M showed high enzyme activity, however, that of mutant CTX-M showed no enzyme activity. Conclusion Ser240 is critical in maintaining the enzyme activity of the extended-spectrum β-lactamase CTX-M.
出处
《中国抗生素杂志》
CAS
CSCD
北大核心
2008年第4期232-234,246,共4页
Chinese Journal of Antibiotics
基金
四川省科技厅应用基础研究项目(2006J13-030)
四川省教育厅重点实验室专项基金资助项目(2006ZD021)
