CMY-39新基因亚型AmpC β-内酰胺酶的特性研究

Characterization of the AmpC β-lactamase of Citrobacter freundii CMY-39 genotype

目的对弗劳地枸橼酸杆菌所产CMY-39新基因亚型AmpCβ-内酰胺酶进行酶特性研究。方法 pET-32a(+)/CMY-39表达质粒在BL21(DE3)中诱导表达,进行SDS-PAGE电泳和Western blotting鉴定酶蛋白的表达;原菌株和重组表达菌株进行药敏试验,并提取重组表达菌株的CMY-39酶蛋白进行AmpC酶三维试验和酶动力学检测。结果 pET-32a(+)/CMY-39在大肠埃希菌中大量表达,蛋白相对分子质量约为60000,用特异抗体经Western检测该条带为阳性;AmpC酶三维试验为阳性;CMY型重组菌株药敏试验结果与原菌株相比,对青霉素类和头孢西丁具有明显的水解作用,第三、四代头孢菌素对之较为稳定,动力学结果与酶稳定性结果基本一致。结论证实重组菌株pET-32a(+)/CMY-39能高效表达CMY-39酶蛋白,表达CMY型重组菌株与原菌株比较,对β-内酰胺类抗生素的敏感性和稳定性有所增加,亲和力下降。

Objective To characterize the β-lactamase from Citrobacter freundii CMY-39 type.Methods The blaCMY-39 was amplified by PCR from pGEM-T /CMY-39.The blaCMY was cloned into pET-32a（＋） vector and trasformed into E.coli BL21（DE3）.The expressed protein was detected by SDS-PAGE and Western blotting.Three-dimensional test,stability and kinetic parameters of the recombinant β-lactamase were performed.The drug resistance of Citrobacter freundii and its recombinant strain were alsoexamined.Results The blaCMY-39 can be expressed in E.coli BL21（DE3）,with molecular weight of 60 000 estimated by SDS-PAGE and Western blotting.The three-dimensional test of AmpC in the recombinant strain was positive.The recombinant strain＇s resistance to antibiotic was decreased compared with Citrobacter freundii.Conclusion CMY-39 type AmpC β-lactamase had been expressed by prokaryotic recombinant plasmid pET-32a（＋）/CMY-39 successfully.Compared with Citrobacter freundii,the recombinant strain＇s susceptibility to β-lactam antibiotics was increased,while the stability was appetency decreased.