文献类型：期刊文献

中文题名：SMAP-29抗菌肽的原核表达、纯化及活性检测

英文题名：Recombinant Expression,Purification and Antimicrobial Activity of SMAP-29

作者：郑秋实[1];段晨曦[1];陶凤云[1]

第一作者：郑秋实

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：3

起止页码：144-148

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(理68);北京联合大学校级科研项目(zk201008x)

语种：中文

中文关键词：抗菌肽;SMAP-29;原核表达;抗菌活性

外文关键词：Antimicrobial peptide ;SMAP-29 ;Prokaryotic expression ;Antimicrobial activity

摘要：旨在利用基因工程技术表达出有活性的重组SMAP-29抗菌肽。根据大肠杆菌偏好的密码子优化smap-29基因序列,在目标肽序列N端添加肠激酶识别位点,C端添加终止密码子,化学合成基因序列,通过EcoR I和Hind III双酶切位点连接到pET-28a(+)上构建重组表达载体,在大肠杆菌BL21(DE3)中表达重组蛋白,Ni-NTA亲和层析纯化,肠激酶切割后释放出SMAP-29抗菌肽,检测其抗菌活性。结果显示,带有6×His标签及肠激酶识别位点的SMAP-29重组融合蛋白在大肠杆菌中以包涵体形式表达,利用Ni-NTA亲和层析可获得纯化的融合蛋白,肠激酶切割后释放出的SMAP-29重组抗菌肽对大肠杆菌、金黄色葡萄球菌和白念珠菌的最小抑菌浓度依次为10、20和40μmol/L。
It was to express active recombination SMAP-29 antibacterial peptides by genetic engineering technology. Optimization smap-29 gone sequences according to Escherichia coli preference codon usage and adding enterokinase recognition sites at N-terminus and stop eodon at C-terminus of the target peptide sequence. The DNA sequence was synthesized by chemical technology and inserted into the pET-28a （ ＋ ） expression vector through the EcoR Ⅰ and Hind sites. Recombinant protein was expressed in Escherichia coli BL21 （ DE3 ） harboring pET28a-smap29 recombinant vector by IPTG induced, and purified by Ni-NTA affinity chromatography. SMAP-29 peptide was released by enterokinase eleavage of the fusion protein and its antibacterial activity was detected. Results showed SMAP-29 recombinant fusion protein with 6 × His tag and enterokinase recognition sites was expressed in inclusion body form, and the fusion protein can by purified by Ni-NTA affinity chromatography. SMAP-29 peptide was released from fusion protein by enterokinase cleavaging. The minimum inhibition concentration （ MIC ） of SMAP-29 against E. coli, S. aureus and C. cdbicans was 10, 20 and 40μmol/L, respectively.