SMAP-29抗菌肽的原核表达、纯化及活性检测

生物技术通报 ›› 2013, Vol. 0 ›› Issue (3): 144-148.

SMAP-29抗菌肽的原核表达、纯化及活性检测

郑秋实,段晨曦,陶凤云

北京联合大学生物化学工程学院，北京 100023

收稿日期:2012-09-28 修回日期:2013-03-21 出版日期:2013-03-20 发布日期:2013-03-21
作者简介:郑秋实，女，学士，研究方向：生物工程；E-mail ：826055785@qq.com
基金资助:
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）

Recombinant Expression,Purification and Antimicrobial Activity of SMAP-29

Zheng Qiushi, Duan Chenxi, Tao Fengyun

Biochemical Engineering College of Beijing Union University,Beijing 100023）

Received:2012-09-28 Revised:2013-03-21 Published:2013-03-20 Online:2013-03-21

摘要/Abstract

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40 μmol/L。

关键词: 抗菌肽, SMAP-29, 原核表达, 抗菌活性

Abstract: It was to express active recombination SMAP-29 antibacterial peptides by genetic engineering technology. Optimization smap- 29 gene sequences according to Escherichia coli preference codon usage and adding enterokinase recognition sites at N-terminus and stop codon 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 III 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 cleavage 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. albicans was 10, 20 and 40 μmol/L, respectively.

Key words: Antimicrobial peptide, SMAP-29, Prokaryotic expression, Antimicrobial activity

郑秋实,段晨曦,陶凤云. SMAP-29抗菌肽的原核表达、纯化及活性检测[J]. 生物技术通报, 2013, 0(3): 144-148.

Zheng Qiushi, Duan Chenxi, Tao Fengyun. Recombinant Expression,Purification and Antimicrobial Activity of SMAP-29[J]. Biotechnology Bulletin, 2013, 0(3): 144-148.

参考文献

[1] Mahoney MM, Lee AY, Brezinski-Caliguri DJ, et al. Molecular analysis of the sheep cathelin family reveals a novel antimicrobial peptide [J]. FEBS Lett, 1995, 377(3):519-522.
[2] Skerlavaj B, Benincasa M, Risso A, et al. SMAP-29 :a potent antibacterial and antifungal peptide from sheep leukocytes [J]. FEBS Lett, 1999, 463(1-2):58-62.
[3] Travis SM, Anderson NN, Forsyth WR, et al. Bactericidal activity of mammalian cathelicidin-derived peptides [J]. Infect Immun, 2000, 68(5):2748-2755.
[4] Weistroffer PL, Joly S, et al. SMAP29 congeners demonstrate activity against oral bacteria and reduced toxicity against oral keratinocytes [J]. Oral Microbiol Immunol, 2008, 23(2):89-95.
[5] Arzese A, Skerlavaj B, Tomasinsig L, et al. Antimicrobial activity of SMAP-29 against the Bacteroides fragilis group and clostridia [J]. J Antimicrob Chemother, 2003, 52(3):375-381.
[6] Benincasa M, Scocchi M, Pacor S, et al. Fungicidal activity of five cathelicidin peptides against clinically isolated yeasts [J]. J Antimicrob Chemother, 2006, 58(5):950-959.
[7] Saiman L, Tabibi S, et al. Cathelicidin peptides inhibit multiply antibiotic-resistant pathogens from patients with cystic fibrosis [J]. Antimicrob Agents Chemother, 2001, 45(10):2838-2844.
[8] Morassutti C, De Amicis F, Bandiera A, et al. Expression of SMAP- 29 cathelicidin-like peptide in bacterial cells by intein-mediated system [J]. Protein Expr Purif, 2005, 39(2):160-168.
[9] 任耀军, 薄新文, 王新华. 抗菌肽SMAP-29 在毕赤酵母中的表达[J]. 石河子大学学报:自然科学版, 2008(6):695-699.
[10] 肇晓光, 马洪星, 张宇雯, 等. 抗菌肽SMAP-29 衍生物的原核表达及纯化[J]. 哈尔滨医科大学学报, 2008(6):563-567.
[11] Dawson RM, Liu CQ. Analogues of peptide SMAP-29 with comparable antimicrobial potency and reduced cytotoxicity [J]. Int J Antimicrob Agents, 2011, 37(5):432-437.
[12] Neville F, Ivankin A, Konovalov O, et al. A comparative study on the interactions of SMAP-29 with lipid monolayers [J]. Biochim Biophys Acta, 2009, 1798(5):851-860.
[13] Shin SY, Park EJ, Yang ST, et al. Structure-activity analysis of SMAP-29, a sheep leukocytes-derived antimicrobial peptide [J]. Biochem Biophys Res Commun, 2001, 285(4):1046-1051.

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