【摘要】 目的对大肠埃希菌所产CTX-M-14型超广谱β-内酰胺酶(ESBLs)进行基因克隆和重组表达,探讨其特性。方法以产CTX-M-14型超广谱β-内酰胺酶大肠埃希菌12号菌总基因组DNA为模板,PCR扩增CTX-M-14,将其克隆入pUCm-T Vector载体后测定该核苷酸序列;再将基因编码区克隆到原核表达载体pET-28α,构建含CTX-M-14基因的重组表达质粒,转化到大肠埃希菌BL21中进行IPTG诱导表达。SDS-PAGE电泳鉴定表达的酶蛋白后再过Ni-NTA柱纯化。结果PCR扩增出大小为876 bp的基因片段,与GenBank上同类酶的基因序列同源性为100%。大肠埃希菌BL21转化pET-28α/CTX-M-14重组质粒后,ESBLs试验为阳性。此基因能在大肠埃希菌中大量表达,SDS-PAGE电泳显示蛋白分子质量大约为30 KD。结论成功表达重组的CTX-M-14型酶,为进一步做酶动力学及酶的其他分子生物学特性研究奠定基础。更多还原

【Abstract】 Objective To express CTX-M-14 extended-spectrum β-lactamase(ESBLs)in pET28α/BL21 system and research the characteristics of the purified CTX-M-14 ESBL.Method The bla CTX-M-14 was amplified by using PCR method from the E.coli genome.The PCR product was cloned into PUC19-T vector and sequenced.In addition,the cloned coding region of CTX-M-14 was inserted into the expression vector pET-28α to form the recombinant plasmid pET-28α-CTX-M-14 which was then transformed into E.coli BL21 for expression.The protein expressed was detected by SDS-PAGE electrophoresis,then purified with Ni-NTA.Result The PCR product had 876 nucleotides and shared 100% amino acid identity with bla CTX-M-14 that already registered in GenBank.The SDS-PAGE analysis revealed that the expressed recombinant CTX-M-14 accumulated up to 45% of the total bacterial protein and its molecular weight was about 30 KD.The test of ESBLs in the recombinant strain was positive.Conclusion The successful prokaryotic expression of bla CTX-M-14 lays a good foundation for enzymological and molecular biological study of CTX-M-14 extended-spectrum β-lactamase.更多还原