新德里金属β-内酰胺酶-1的表达、纯化与活性测试  

Over-expression, purification and bio-activity testing of New Delhi metallo-β-lactamase-1

作　　者：王伊雪 蒋越 施洋 翟乐 WANG Yi-xue;JIANG Yue;SHI Yang;ZHAI Le(College of Chemistry and Chemical Engineering,Baoji University of Arts and Sciences,Baoji 721013,Shaanxi,China)

机构地区：[1]宝鸡文理学院化学化工学院,陕西宝鸡721013

出　　处：《宝鸡文理学院学报（自然科学版）》2022年第4期21-27,34,共8页Journal of Baoji University of Arts and Sciences(Natural Science Edition)

基　　金：陕西省创新能力支撑计划科技创新团队项目(2022TD-63);陕西省教育厅重点科学研究计划(重点实验室)项目(17JS007)。

摘　　要：目的 通过E.coli BL21(DE3)系统对重组质粒pET26b-NDM-1进行表达,纯化得到新德里金属β-内酰胺酶-1(New Delhi metallo-β-lactamase-1, NDM-1),为新型NDM-1抑制剂的生物活性评价提供物质条件。方法 将pET26b-NDM-1重组质粒转化后,在E.coli BL21(DE3)中进行诱导、表达;通过快速蛋白液相色谱系统,利用含氯化钠缓冲溶液对目标蛋白进行洗脱并以十二烷基硫酸钠聚丙烯酰胺凝胶电泳(Sodium Dodecyl Sulfate Polyacrylamide gel Electrophoresis, SDS-PAGE)检测纯度;利用稳态动力学研究方法,通过测定NDM-1催化头孢唑啉钠水解反应的初速率以及乙二胺四乙酸(Ethylene Diamine Tetraacetic Acid, EDTA)对该反应的半数抑制浓度(Half Maximal Inhibitory Concentration, IC50),验证并评价所制备NDM-1的生物活性。结果 SDS-PAGE及UV-Vis表征结果显示,所制备NDM-1分子量正确,纯度大于95%;稳态酶动力学活性鉴定结果表明,所得NDM-1催化活性良好,水解头孢类β-内酰胺抗生素头孢唑啉钠能力强,速率快,EDTA对该NDM-1催化底物水解反应的IC50=7.803μM。结论 表达、纯化所得金属β-内酰胺酶NDM-1具有优良的催化活性,可用以评价新型NDM-1抑制剂的抑制效果。Purposes-To evaluate the inhibitory activity of novel NDM-1 inhibitors by over-expressing NDM-1 gene from recombinant plasmid pET26b-NDM-1 in E. coli BL21(DE3) and obtain the purified New Delhi metallo-β-lactamase-1(NDM-1) through specific purification process. Methods-The plasmid pET26b-NDM-1 was transformed and induced in E. coli BL21(DE3). The harvested bacteria cells were collected by centrifugation and lysed by ultrasonication. Cell debris were separated by centrifugation. The cleared supernatant was dialyzed and loaded onto an equilibrated Q-Sepharose column eluted with buffer containing NaCl. Fractions containing NDM-1 were pooled and concentrated with an Amicon ultrafiltration cell equipped with an YM-10 membrane. Protein purity was ascertained by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The steady-state enzyme inhibition kinetics experiment was carried out through Agilent 8454 UV-Vis spectrophotometer. The bioactivity of NDM-1 was evaluated by measuring the initial reaction rate of cefazolin hydrolysis by NDM-1. The inhibitory activity of EDTA against NDM-1 was also verified by IC50assay. Results-SDS-PAGE and UV-Vis data indicated that the obtained NDM-1 displayed fine protein purity. The steady-state enzyme inhibition kinetics experiments revealed the NDM-1 exhibited efficient catalytic activity toward cefazolin. The inhibitory activity of EDTA against NDM-1 was confirmed(IC50= 7.803 μM). Conclusion-The purified metallo-β-lactamase-1 NDM-1 showed excellent catalytic activity, which can be used to evaluate the biological activity of novel NDM-1 inhibitors.

关 键 词：细菌耐药性 新德里金属β-内酰胺酶-1 Β-内酰胺抗生素 稳态动力学 

分 类 号：O622.5[理学—有机化学]