摘要
目的对肺炎克雷伯菌所产CTX-M-3型超广谱β-内酰胺酶(extended-spectrum β-lactamase,ESBLs)进行基因重组表达及纯化。方法以产CTX-M-3型超广谱β-内酰胺酶肺炎克雷伯菌总基因组DNA为模板PCR扩增CTX-M-3,将其克隆入pET-28a(+)载体,重组质粒转入大肠埃希菌ER2566中表达,表达产物经过Ni-琼脂糖凝胶柱纯化,用SDS-PAGE电泳和Western-blot鉴定纯化蛋白。结果PCR扩增出大小为876bp的基因片段,与GenBank上同类酶的基因序列同源性为100%。此基因能在大肠埃希菌ER2566中大量表达,蛋白分子质量大约为32KD得到SDS-PAGE电泳和Western-blot的证实,与理论值相符。结论成功表达及纯化CTX-M-3型超广谱β-内酰胺酶,为进一步酶生物学特性及酶的抗体制备研究奠定了基础。
Objective To express and purify CTX-M-3 extended-spectrum β-lactamase from Klebsiella pneumoniae. Methods The bla CTX-M-3 was amplified by PCR and sequenced after being subcloned into pGEM-T vector. The blaCTX-M-3 was cloned into pET-28a（＋） vector ,and expressed in E.coli 2 566,and then purified with Ni-NTA. The purified protein was identifid by SDS-PAGE and Western-blot. Results Sequence of blaCTX-M-3 shared 100% amino acid identity with blaCTX-M-3 that already registered in GenBank. The blaCTX-M-3 could be expressed in E.coli 2566 ,purified with.Ni-NTA,its molecular weight was about 32KD. The recombinant protein was expressed and confirmed by SDS-PAGE and Western-blot. Conclusions The successful prokaryotic expression and purification of blaCTX-M-3 has laid a foundation for carrying out study on molecular biology and preparation of antibody of CTX-M-3 extended-spectrum β-lactamase.
出处
《中国热带医学》
CAS
2010年第2期137-138,161,共3页
China Tropical Medicine
基金
广州市医药卫生科技(2007-YB-156)
广州市医药卫生科技(2008-YB-149)
