新德里金属β-内酰胺酶的表达及活性

The study on expression and activity of New Delhi metal-β-lactamase-1

利用前期构建的重组表达菌Tb1(pMAL-p2X::NDM-1),以温度、时间和IPTG浓度建立三因素三水平的正交试验,对该重组蛋白诱导表达条件进行优化。用Amylose树脂对重组蛋白进行纯化,Xa因子进行切割,MALDITOF-MS鉴定蛋白。测定重组表达菌对亚胺培南的最小抑菌浓度,分光光度法测定蛋白酶的活性及EDTA抑制作用。结果显示,37℃,IPTG 0.3mmol/L,诱导8h为MBP-NDM-1融合表达的最优条件,表达量占菌体蛋白的39.2%,MALDI-TOF-MS显示重组蛋白相对分子质量为71 200.811,经Xa切割后目的蛋白相对分子质量为21 053.324,重组表达菌对亚胺培南的MIC为0.064g/L,重组蛋白对亚胺培南的米氏常数Km=69.347μmol/L,25μmol/L的EDTA可以抑制重组蛋白的酶活性。本试验对NDM-1蛋白酶活性进行了研究,为后续NDM-1酶抑制剂的研究奠定了基础。

On the basis of the recombinant plasmid constructed by our laboratory, a three factors （including culture temperature,inducing time and concentration of IPTG） orthogonal experiment was established to study the optimal expression conditions of recombinant protein. The fusion pro tein was purified using the amylase column and cleaved by Factor Xa. The protein was identified by MALDI-TOF-MS. The MICs of recombinant strain TBI（pMAL-p2X： ： NDM-1） resistant to imi penem was determined by serial 2-fold dilution method. The enzymatic activity and inhibition of EDTA were performed using imipenem as substrate. The result is that the optimal temperature, inducing time and concentration of IPTG of the fusion expression were 37℃ ,8 h and 0.3 mmol/L, respectively. The fusion protein accounted for 39.2% of the total bacteria protein. MALDI-TOF- / MS showed that the molecular weight of the fusion protein and protein cleaved by Factor Xa were 71 200. 811 and 21 053. 324, respectively. The MIC of imipenem for Tbl（pMAL p2X： ：NDM-1） was 0. 064 g/L. The Km values of the fusion protein for the imipenem was 69. 347 /μmol/L. EDTA with a value of 25 /μmol/L displayed an inhibitory effect on the fusion protein enzymatic activity in vitro . We studied the optimal expression, conditions and the enzymatic activity of NDM-1 which provided the foundation for further study on the development of inhibitors to NDM-1.