鸡福氏志贺菌SHV-12型ESBLs基因的扩增、原核表达及酶特性研究  

PCR Amplication,Prokaryote Expression of SHV-12 Type Extend-spectrum β-lactamase Produced by Shigella Isolated from Fowl and Its Related Characteristics

作　　者：孙亚伟[1] 胡功政[1] 陈红英[1] 刘建华[1] 苑丽[1] 邓立新[1] 袁业友[1] 

机构地区：[1]河南农业大学牧医工程学院,郑州450002

出　　处：《华中农业大学学报》2009年第1期65-69,共5页Journal of Huazhong Agricultural University

基　　金：国家自然科学基金项目(30471307);河南农业大学创新基金项目(2007)资助

摘　　要：对5株鸡福氏志贺菌产SHV-12型ESBLs基因进行重组表达并优化表达条件,观察了表达蛋白的底物水解特性和抑制剂的抑酶特性。结果表明:构建的重组质粒经EcoRⅠ和XholⅠ双酶切后,可切下目的条带。重组质粒转化BL21后所表达的重组蛋白有超广谱β-内酰胺酶活性。SHV-12原核表达的最佳条件是IPTG浓度为0.5 mmol/L、温度37℃、诱导时间5 h;SHV-12型ESBLs亲和力最强的底物是头孢噻呋钠,Km值为1.29μmol,较难水解的底物是头孢曲松钠,Km值为24.13μmol;它唑巴坦、舒巴坦对头孢曲松钠的抑酶保护率分别为21.01%、25.51%,对头孢噻呋钠的抑酶保护率分别为34.99%、35.49%。SHV-12 type extended-spectrum β-lactamase genes produced by 5 shigella isolates fromfowl was cloned and expressed under optimized condition and related characteristics of the expressed proteins were studied, The results showed that the taget fragment was obtained after the constructed recombinant plasmids was digested by EcoR I and Xhol I . The constructed recombinant plasmids expressed recombinant proteins which possessed responding ESBLs activities. The optimized conditions of expressing recombinant strains S-BL21 was： IPTG concenration was 0. 5 mmol/L, the induced temperature was 37 ℃ ,the induced time was 5 h. The SHV-12 type ESBLs has power affinity to ceftifur sodium with Km value of 1.29 /μmol and has more difficulty to hydrolyse ceftriaxone with Km value of 24. 13 /μmol. The inhibition rate of tazobactam and sulbactam on hydrolyzing eeftriaxone and ceftiofur of SHV-12 type ESBLs was 21.01%,25.51%,34.99%,and 35.49% respectively.

关 键 词：福氏志贺菌 SHV-12型ESBLs 原核表达 酶特性 

分 类 号：Q785[生物学—分子生物学] Q786