CAJ | 학술논문

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.
목적 대량주불로지구연산간균소산CMY형AmpC매진행기인극륭、서렬분석화중조표체재체적구건.방법 이산CMY형AmpC매적불로지구연산간균30、31호총기인조DNA위모판,PCR확증CMY기인,장기극륭인pGEM-T재체후측정해핵감산서렬.30、31호균여수체균진행질립접합실험,구건pBV220-CMY중조표체재체.대원균주화중조균주진행AmpC매검측.결과 PCR확증출대소위1146 bp적기인편단,여GenBank상다충CMY아형적기인서렬동원성위97%.질립접합실험증실질립상함CMY기인,위가전이질립.삼유실험결과현시30、31호균화중조균주소산적매균능수해두포서정.결론 30、31호균소산적CMY형AmpC매위CMY신기인아형.성공구건중조표체재체pBV220-CMY,위하일보매적표체화순화제공료의거.
Objective 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.