摘要目的 对两株弗劳地枸橼酸杆菌所产CMY型AmpC酶进行基因克隆、序列分析和重组表达载体的构建.方法 以产CMY型AmpC酶的弗劳地枸橼酸杆菌30、31号总基因组DNA为模板,PCR扩增CMY基因,将其克隆人pGEM-T载体后测定该核苷酸序列.30、31号菌与受体菌进行质粒接合实验,构建pBV220-CMY重组表达载体.对原菌株和重组菌株进行AmpC酶检测.结果 PCR扩增出大小为1146 bp的基因片段,与GenBank上多种CMY亚型的基因序列同源性为97%.质粒接合实验证实质粒上含CMY基因,为可转移质粒.三维实验结果显示30、31号菌和重组菌株所产的酶均能水解头孢西丁.结论 30、31号菌所产的CMY型AmpC酶为CMY新基因亚型.成功构建重组表达载体pBV220-CMY,为下一步酶的表达和纯化提供了依据.

abstractsObjective To carry out gene cloning, sequence analysis and recombinant expression vector construction of CMY-type AmpC β-lactamase from two strains of Citrobacterfreundii. Methods Total genomic DNA from Citrobacterfreundii strain 30, 31 which produced CMY-type AmpC β-1aetamase acted as templar, the blaCMY was amplified by PCR. Nucleotide sequence measure was performed after blaCMY had being cloned in pGEM-T vector. Plasmid conjugation test were examined between strain 30, 31 and E.coli HBIO1Rif. And then the blaCMY was cloned into pBV220 vector. AmpC induce tests of strain 30, 31 and recombinant strain were detected. Results 1146 bp DNA fragment of CMY-type AmpC β-lactamase by PCR showed 97% amino acid identity with blaCMY which already registered in GenBank. Plasmid conjugation test proved blaCMY located in plasmid and plasmid could conjugate. The results of three-dimension test showed AmpC β-1actamase from strain 30, 31 and recombinant strain could hydrolyze cefoxitin. Conclusion The blaCMY from strain 30, 31 was a novel gene subtype of CMY- type AmpC β- laetamase. Recombinant expression vector pBV220/CMY is successfully constructed, which provides good evidence for further study of expression and purification of blaCMY.