CAJ | 학술논문

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Nde I、Xho I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Nde I、Xho I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.
목적 대래자폐염극뢰백균림상균주적LEN-5형β-내선알매진행기인극륭화중조표체.방법 종상응림상균주제취질립DNA;이질립위모판,PCR확증LEN-5기인,확증산물경Nde I、Xho I매절후련접지pET-26b (+)표체재체,중조질립경DNA측서학증후,전입대장간균BL21(DE3)진행유도표체.초성파쇄법제취단백표체산물,두포초새분검측기활성,병검측단백적등전점(pI).결과 PCR확증획득879bp적산물,DNA서렬현시해편단서렬여목적 서렬완전일치.중조표체재체경Nde I、Xho I매절급DNA측서후표명,목적 기인이성공접입표체재체,표체산물경두포초새분검측현시구유β-내선알매활성,현시재체[pET-26b(+)/LEN-5]구건성공.표체단백적등전점위7.6.결론 β-내선알매LEN-5기인재원핵세포중완성료중조화표체,위진일보주매동역학급매적기타분자생물학특성연구전정기출.
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.