【摘要】 目的构建SHV-59型β-内酰胺酶的表达载体。方法抽提菌株的质粒,应用PCR扩增SHV-59基因全长编码序列,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879 bp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,表明载体[pET-26b(+)/SHV-59]构建成功。目的等电点为7.6。结论β-内酰胺酶SHV-59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。更多还原

【Abstract】 Objective To express SHV-59 β-lactamase in pET-26b(+)/BL21(DE3)system. Method Plasmid in the strain was extracted;PCR was used for amplification of SHV-59 gene.After being digested with Nde I and Xho I,the SHV-59 gene was cloned into pET-26b(+) vector.Before transformed into E.coli BL21(DE3),the SHV-59 gene in recombinant plasmid was confirmed by digestion and DNA sequencing.SHV-59 β-lactamase was expressed after induced by IPTG.Protein extraction was processed by Ultrasonic,and the protein activity was detected by Nitrocefin.The protein isoelectric point(PI) was determined by isoelectric focus.Result A 879 bp amplified product was obtained by PCR.Digestion with Nde I,Xho I and DNA sequencing of the recombinant vector showed that the target gene had been successfully cloned into the expression vector.Mitrocefin test showed that the recombinant protein had β-lactamase activity,indicating that the expression vector [pET-26b(+)/SHV-59] was constructed successfully.The PI of SHV-59 was 7.6.Conclusion SHV-59 gene can be expressed in prokaryote cell,which laid a foundation for further analysis of this novel β-lactamase.更多还原