鸡福氏志贺菌产超广谱β-内酰胺酶SHV-12和TEM-1V基因的重组表达及酶特性  

Cloning and expression of SHV-12 and TEM-1V type extended-spectrum β-lactamase genes produced by chicken shigella and their related characteristics

作　　者：孙亚伟[1] 胡功政[1] 陈红英[1] 邓立新[1] 刘建华[1] 潘玉善[1] 许兰菊[1] 李胜利[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002

出　　处：《中国兽医学报》2009年第5期566-571,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30471307)

摘　　要：通过PCR扩增获得鸡福氏志贺菌产超广谱β-内酰胺酶SHV-12和TEM-1V型ESBLs基因片段,定向克隆入质粒载体构建重组质粒,酶切和DNA测序鉴定后转化受体菌BL21,用SDS-PAGE电泳观察目的基因在重组菌中的表达,用双紫外分光光度仪测定表达蛋白的底物水解特性和抑制剂的抑酶作用。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;DNA测序发现插入片段与SHV-12和TEM-1V ESBLs基因序列完全一致。重组质粒转化BL21所表达的重组蛋白有超广谱β-内酰胺酶活性。重组菌S-BL21、T-BL21的最优表达条件分别是IPTG浓度0.5、0.8mmol/L,诱导温度37℃、37℃,诱导时间5、4h。SHV-12和TEM-1V型ESBLs能水解青霉素G、氨苄西林、阿莫西林、头孢噻呋、头孢曲松和头孢噻肟;对头孢噻呋钠的Km值最小,分别为4.9和1.29;对头孢曲松钠的Km值最大,分别为53.43和24.13;抑制剂舒巴坦钠、它唑巴坦钠对SHV-12和TEM-1V型ESBLs水解抗生素有不同程度的抑制作用。SHV-12 and TEM-1V type extended-spectrum β-1actamase（ESBLs） genes produced by shigella isolated from fowl were cloned and expressed under optimized condition, and related enzyme characteristics of the expressed proteins were studied. The SHV-12 and TEM-1V ESBLs gene fragments were obtained by using polymerase chain reaction. After digestion by restriction enzyme EcoR Ⅰ and Xhol Ⅰ , the gene fragments were inserted into plasmid vector PET-28a to constructe the recombinant ptasmid. The recombinant plasmid was transformed to BL21 receptor. Gene expression was analaysed by using SDS-PAGE. The characteristics of antibiotic hydrolysis and inhibition of 13-1actamase inhibitors on SHV-12 and TEM-1V ESBLs were observed with ultraviolet spectrophotometer. The results revealed that the constructed recombinant plasmids expressded recombinant proteins which possessed responding ESBLs activities. The optimized conditions of expressing recombinant strains S-BL21 and T-BL21 were 0.5,0.8 mmol/L IPTG;37℃ and 37℃ induced temperatures;and 5 h and 4 h induced times,respectively. SHV-12 and TEM-1V type ESBLs may hydrolyze penicillinG, ampicillin, amoxicillin, ceftiofur, ceftriaxone, ceftriaxone and cefotaxime,Km values to ceftiofur were 4. 9 and 1. 29, respectively, to ceftriaxone 53. 43 and 24. 13, respectively. Tazobactam and sulbactam inhibited hydrolysis of SHV-12 and TEM-1V type ESBLs on β-1actams to various extends.

关 键 词：鸡福氏志贺菌 超广谱Β-内酰胺酶 SHV-12 TEM-1V 

分 类 号：S852.61[农业科学—基础兽医学]