SHV-59型β-内酰胺酶的克隆与表达  

Cloning and expression of SHV-59 β-lactamase gene

作　　者：卢月梅[1,2] 张阮章[1,2] 王沙燕[1,2] 胡玉华[1,2] 穆雪鵾 何林[1,2] 陈升汶[1,2] 

机构地区：[1]深圳市人民医院 [2]暨南大学第二临床学院检验科,广东深圳518020

出　　处：《中国微生态学杂志》2009年第9期825-826,832,共3页Chinese Journal of Microecology

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。Objective To express SHV-59 β-lactamase in pET-26b （ ＋ ）/BL21 （DE3） system. Method Has- mid 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 Nitrocef＇m. The protein i- soelectric point（PI） was determined by isoelectric focus. Result A 879 bp amplified product was obtained by PCR. Diges- tion 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.

关 键 词：Β-内酰胺酶 原核表达 等电聚焦电泳 

分 类 号：R978.11[医药卫生—药品]