OKP-B-13型β-内酰胺酶的克隆与表达

Cloning and expression of OKP-B-13 β-lactamase gene

目的构建OKP-B-13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OKP-B-13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,显示载体[pET-26b(+)/OKP-B-13]构建成功。目的等电点为7.1。结论β-内酰胺酶OKP-B-13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。

Objective To express OKP-B-13 β-lactamase in pET26b（＋）/BL21（DE3）system.Method The plasmid in the strain was extracted;PCR was used for amplification of OKP-B-13 gene.After digested with Nde I and Xho I,OKP-B-13 gene was cloned into pET-26b（＋） vector.Before transformed into E.coli.BL21（DE3）,the OKP-B-13 gene in recombinant plasmid was confirmed by digestion and DNA sequencing.OKP-B-13 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.The result of digestion test and DNA sequence of the recombinant vector verified that the target gene had been successfully cloned into the expression vector.The recombinant protein was shown to have β-lactamase activity by Nitrocefin test,indicating that the expression vector[pET-26b（＋）/OKP-B-13] was constructed successfully.The pI of OKP-B-13 was 7.1.Conclusion OKP-B-13 gene can be expressed in prokaryote cell,which may provide foundation for further analysis of this novel β-lactamase.