文献类型：期刊文献

中文题名：中国林蛙核糖核酸酶Rdrlec的原核表达及抗菌活性检测

英文题名：Prokaryotic Expression and Antimicrobial Activity of a Novel Ribonuclease Rdrlec from Rana dybowskii

作者：尹雪薇[1];彭艳丽[1];冉帅[1];陶凤云[1]

第一作者：尹雪薇

机构：[1]北京联合大学生物化学工程学院

第一机构：北京联合大学生物化学工程学院

年份：2013

期号：5

起止页码：93-98

中文期刊名：生物技术通报

外文期刊名：Biotechnology Bulletin

收录：CSTPCD;;北大核心:【北大核心2011】；CSCD:【CSCD_E2013_2014】；

基金：北京联合大学"启明星"大学生科技创新项目;北京联合大学校级科研项目(zk201008x)

语种：中文

中文关键词：中国林蛙;核糖核酸酶;Rdrlec;原核表达;抗菌活性

外文关键词：Rana dybowskii Ribonuclease Rdrlec Prokaryotic expression Antimicrobial activity

摘要：Rdrlec是从中国林蛙基因组中克隆得到的核糖核酸酶新基因(GenBank登录号EU384704),其生物学活性仍未知。为了探索其编码的蛋白产物的生物学活性,将此基因按照大肠杆菌偏好的密码子进行优化后,人工合成基因,通过EcoR I和HindIII限制酶位点定向克隆到pET-28a(+)中构建重组表达载体,转化到大肠杆菌BL21(DE3)中,在34℃以0.5 mmol/L IPTG诱导表达出包涵体形式的融合蛋白,通过包涵体复性、肠激酶切割、Ni-NTA亲和层析纯化等步骤获得了具有酶活性的野生型Rdrlec重组蛋白,并检测到其对大肠杆菌和金黄色葡萄球菌具有显著的抗菌活性。
Rdrlec is a novel RNase gene form Rana dybowskii（GenBank accession No.EU384704）and its biological function has not been identified.In order to explore the biological activity of its encoded protein,the Rdrlec gene was adjusted according to Escherichia colicodon bias without changing its amino acids.The synthetic gene was inserted to the pET-28a（＋）expression vector through the EcoR I and Hind III site,and the resulting recombination expression plasmid was named pET28-Rdrlec and transferred into Escherichia coli BL21（DE3）strains.After induced with 0.5 mmol/L IPTG at 34℃ for 6 h,the fusion protein was found expressed mainly in inclusion body form.After a series of steps including refolding,enterokinase cutting and Ni-NTA affinity chromatographic purification,the wild type Rdrlec recombinant protein was obtained,and it showed a single band on SDS-PAGE.It showed enzymatic activity to degrade RNA into nucleotides,which suggests that this molecule has formed the correct spatial structure.The recombinant Rdrlec shows significant antibacterial activity against Escherichia coli and Staphylococcus aureus.