嗜麦芽窄食单胞菌所产金属β-内酰胺酶基因的克隆及表达

Cloning and expression of metallo beta-lactamase encoding gene found in a clinical isolate of Stenotrophomonas maltophilia

目的 探讨嗜麦芽窄食单胞菌所产金属 β 内酰胺酶的酶学特性及分子生物学特征。方法 以MBL E试验筛查到的一株产金属 β 内酰胺酶 (MBL)嗜麦芽窄食单胞菌 75 0为研究对象 ;MBL的等电点、对酶抑制剂的酶稳定性及抗生素的水解率按常规测定 ;抽提细菌染色体、PCR扩增blaMBL全基因并进行测序 ;构建blaMBL pET32a( + )重组体 ,并在大肠杆菌BL2 1(DE3)中进行表达。结果 菌株 75 0仅产一种 β 内酰胺酶 ,其pI为 6 6 ;以对亚胺培南的水解率为 10 0 %计 ,对青霉素、拉氧头孢、头孢他啶、氨曲南的相对水解率分别依次为 14 2 0 %、2 7%、9%和 1% ,酶活性可被EDTA抑制 ,但不受克拉维酸、舒巴坦、他唑巴坦等 β 内酰胺酶抑制剂影响。染色体DNA模板的PCR结果显示 ,酶全基因全长为 86 7bp ,经序列分析和同源性比较 ,与已报道的金属酶L1的编码基因blaS(注册号AJ2 916 72 )和blaL1(AF0 10 2 82 )的核苷酸同源性均为 99 31% ,推定的氨基酸序列同源性分别为 99 31%和 98 96 %。大肠杆菌BL2 1(DE3)转化含酶基因的blaMBL pET32a( + )重组质粒后 ,MBL E试验为产MBL阳性 ,对亚胺培南和头孢他啶的MIC分别为 12mg/L和 2mg/L ,对亚胺培南的MIC较母株低 10倍左右 ;SDS PAGE分析发现 ,融合基因表达的蛋白为 4 8kDa 。

Objective To learn Molecular characterization of metallo beta-lactamase(MBL) found in a clinical isolate of Stenotrophomonas maltophilia and confirm the role of MBL played in the antimicrobial-resisitance of S. maltophilia by sequencing the encoding genes of the metallo beta-lactamase and construct the prokaryotic expression vector carrying the MBL gene and expressed in E.coli BL 21 .Methods The blaMBL gene was amplified by PCR and cloned into pMD18-T plasmid. The recombination was subcloned into pET32a(+) plasmid and expressed in E.coli BL 21 . The susceptibility between expression vectors and strain 750 to antibiotics were compared.Results The 867 bp DNA fragment of MBL encoding gene was amplified from the strain 750 by PCR and sequenced. The gene was 99.31% homologous to blaS and blaL1 of MBL L1. After being transformed into the E.coli BL 21 and induced with lmM IPTG, a recombinant protein of about 48 kDa was expressed in the pET32a(+)-blaMBL stsytem. The susceptibility of pET32a(+)- blaMBL system and strain 750 showed MIC 12 mg/L and 128 mg/L to imipenem and MIC 2mg/L and 2mg/L to ceftazidime, respectively. Conclusion The MBL produced by strain 750 was similar to the that in strain ULA511. The difference of MIC to imipenem between wild strain and E.coli BL 21 transformant indicated that other unclear mechanism involving in imipenem resistance in the strain.