LEN-5型β-内酰胺酶基因的克隆及原核表达

Cloning and prokaryotic expression of LEN-5 β-lactamase gene

目的对来自肺炎克雷伯菌临床菌株的LEN-5型β-内酰胺酶进行基因克隆和重组表达。方法从相应临床菌株提取质粒DNA;以质粒为模板,PCR扩增LEN-5基因,扩增产物经NdeI、XhoI酶切后连接至pET-26b(+)表达载体,重组质粒经DNA测序确证后,转入大肠杆菌BL21(DE3)进行诱导表达。超声破碎法提取蛋白表达产物,头孢硝噻吩检测其活性,并检测蛋白的等电点(pI)。结果PCR扩增获得879bp的产物,DNA序列显示该片断序列与目的序列完全一致。重组表达载体经NdeI、XhoI酶切及DNA测序后表明,目的基因已成功接入表达载体,表达产物经头孢硝噻吩检测显示具有β-内酰胺酶活性,显示载体〔pET-26b(+)/LEN-5〕构建成功。表达蛋白的等电点为7.6。结论β-内酰胺酶LEN-5基因在原核细胞中完成了重组和表达,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。

In order to express the gene of LEN-5 β-lactamase from a Klebsiella pneumoniae strain,plasmids in the strain were extracted and an 879bp product of LEN-5 gene was obtained with PCR.After being digested with Nde I and Xho I,LEN-5 gene was cloned into pET-26b（＋） vector.Then it was confirmed by digestion and DNA sequencing in recombinant plasmid before transformed into E.coli BL21（DE3）.After inducing by IPTG,LEN-5 β-lactamase was expressed.Protein extraction was processed by ultrasonic and protein activity was detected by nitrocefin.The isoelectric focusing electrophoresis showed a pI of 7.6.These results indicated that the LEN-5 gene has been cloned and expressed in prokaryote cell successfully.