CTX-M-14型超广谱β-内酰胺酶的序列分析与原核表达

Sequence analysis and prokaryotic expression of the CTX-M-14 extended-spectrum β-lactamase

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。

Objective To express CTX-M-14 extended-spectrum β-1aetamase （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 recombinam plasmid pET-28α- CTX-M-14 which was then transformed into E. coli BI21 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 recom- binant 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-1aetamase.