CAJ | 학술논문

目的构建新型β-内酰胺酶CTX-M-38的表达载体.方法应用PCR扩增CTX-M-38基因全长编码序列,经Nde I、XhoⅠ酶切后连接至pET-26b(+)表达载体,重组质粒经酶切及DNA测序确证后,转入大肠埃希菌BL21(DE3),IPTG诱导表达.超声破碎法提取表达蛋白产物,检测其活性,等电聚焦电泳检测蛋白的等电点(pI).结果 PCR扩增获得894bp的产物,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,重组菌的粗提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(+)/CTX-M-38]构建成功.蛋白pI为8.4.结论 β-内酰胺酶CTX-M-38在原核表达细胞中实验了基因重组表达,为进一步分析酶的特性提供条件.
목적 구건신형β-내선알매CTX-M-38적표체재체.방법 응용PCR확증CTX-M-38기인전장편마서렬,경Nde I、XhoⅠ매절후련접지pET-26b(+)표체재체,중조질립경매절급DNA측서학증후,전입대장애희균BL21(DE3),IPTG유도표체.초성파쇄법제취표체단백산물,검측기활성,등전취초전영검측단백적등전점(pI).결과 PCR확증획득894bp적산물,중조표체재체경Nde I.Xho I매절급DNA측서후표명,목적 기인이성공접입표체재체,중조균적조제물경두포초새분검측현시구유β-내선알매활성,현시재체[pET-26b(+)/CTX-M-38]구건성공.단백pI위8.4.결론 β-내선알매CTX-M-38재원핵표체세포중실험료기인중조표체,위진일보분석매적특성제공조건.
Objective To express CTX-M-38 β-lactamase in pET26b(+)/BL21(DE3)system.Methods Plasmid in strain Was extracted,PCR was used for amplification of CTX-M-38 gene.After been digested with NdeI and Xho I,CTX-M-38 gene Was cloned into pET-26b(+)vector.Before transformed into E.coli.BL21(DE3),CTX-M-38 gene in recombinant plasmid was confirmed by digestion and DNA sequencing.CTX-M-38 β-lactamase was expressed after induced by IPTG,protein extraction processed by ultrasonic,and the protein activity was detected by nitrocefin,The protein isoelectric point(pI)Was determined by isoelectric focus.Results A 879bp amplified product Was obtain by PCR.Digestion with NdeI.XhoI and DNA sequencing for the recombinant vector showed that the target gene has been successfully cloned into the expression vector.the recombinant protein was showed to be with β-lactamase activity by nitrocefin test,indicating expression vector[pET-26b(+)/CTX-M-38]Was constructed successfully.The pI of CTX-M-38 is 8.4.Conclusions CTX-M-38 gene can be expressed in prokaryote cell.and this may be basis for further analysis of this novel β-lactamase.