VEB-3型与VEB-1型超广谱β-内酰胺酶生化特性的比较

Comparison of biochemical characteristics between VEB-3 and VEB-1 extended-spectrum beta-lactamase

目的研究VEB-3型超广谱β-内酰胺酶(ESBL)的生化特性,并与VEB-1型ESBL进行比较。方法将blaVEB-3和blaVEB-1基因连接至pET28a载体上,并用重组质粒转化大肠埃希菌BL21进行克隆表达,分别挑选blaVEB-3或blaVEB-1阳性表达的菌落测定β-内酰胺类抗生素对其最低抑菌浓度(MIC),同时提取酶蛋白进行等电点和酶动力学分析。结果VEB-3和VEB-1型ESBL具有基本一致MIC和相同的等电点(pI=7.45),但两者的酶动力学性质存在一定的差异:对头孢他啶和头孢噻肟,VEB-3较VEB-1表现出更高的亲和力,两者Km相差一倍左右;对头孢噻吩,VEB-1比VEB-3更具亲和力;而对阿莫西林,VEB-1型ESBL表现出很高的亲和力,而VEB-3仅有低的亲和力,两者Km相差20多倍。结论VEB-3和VEB-1型ESBL具有相同的等电点,不同的动力学参数,blaVEB-1基因中Leu56→Phe突变可能导致了两者酶动力学性质的差异。

Objective Biochemical characterization of VEB-3 extended-spectrum beta-lactamase （ESBL） and to compare it with VEB-1 ESBL. Methods blaVEB-3 and blaVEB-1 genes were ligated with pEq28a vector respectively. Reeombinated plamids blaVEB-3/pET28a and blaVEB-1/pET28a were eleetroporated into E. coli BL21 cells. The recombinant strains were identified by sequence analysis. MIC of the recombinant strains were determined by E test method. Meanwhile, the beta-lactamases were obtained from recombinant strains and then the iseelectric points（pI） and kinetic parameters were determined. Results The recombinant strain expressing VEB-3 ESBL had same MIC with the recombinant strain expressing VEB-1 ESBL. VEB-3 and VEB-1 ESBL has the same pI（7.45）. But the kinetic parameters were significantly different between them： VEB-3 ESBL showed a higher affinity to ceftazidime and cefotaxime. Instead, the Km value for cephalothin of VEB-1 was lower than that of VEB- 3. The Km value for arnoxicillin of VEB-3 was about twenty-three times of that of VEB-1. Conclusion Between VEB-1 and VEB-3 ESBL, there are the same pI and the different kinetics that could arise from the mutation of Leu56→Phe in VEB-1 gene.