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棘胸蛙抗菌肽Spinosan-C的串联表达与活性检测
引用本文:刘悦,詹忠根,朱兵,郑荣泉,程宏毅,聂作明.棘胸蛙抗菌肽Spinosan-C的串联表达与活性检测[J].生物工程学报,2018,34(1):132-139.
作者姓名:刘悦  詹忠根  朱兵  郑荣泉  程宏毅  聂作明
作者单位:1 浙江经贸职业技术学院,浙江 杭州 310018,1 浙江经贸职业技术学院,浙江 杭州 310018,2 浙江理工大学 生命科学学院,浙江 杭州 310018,3 浙江师范大学 生态研究所,浙江 金华 321004,3 浙江师范大学 生态研究所,浙江 金华 321004,2 浙江理工大学 生命科学学院,浙江 杭州 310018
基金项目:浙江省公益计划项目 (No. 2014C32071),金华市科技计划重点项目 (No. 2013-2-1008)资助。
摘    要:为克服抗菌肽易被蛋白酶降解及对宿主大肠杆菌的杀伤作用,并进一步提高大肠杆菌系统的表达能力,以棘胸蛙Paa spinosa抗菌肽Spinosan-C为研究对象,按照大肠杆菌密码子利用频率进行密码子优化,设计合成8拷贝的串联8×Spinosan-C基因,将合成的串联基因克隆到大肠杆菌表达载体p ET-28a,利用大肠杆菌感受态细胞Rosetta进行原核表达,获得高效表达的串联8×Spinosan-C重组蛋白,用甲酸专一性切割得到抗菌肽Spinosan-C单体。体外抑菌试验表明,切割后的抗菌肽Spinosan-C单体对测试菌生长具有抑制作用,为蛙类抗菌肽的规模化制备提供了参考。

关 键 词:棘胸蛙抗菌肽,Spinosan-C,串联表达,专一性切割,抗菌活性
收稿时间:2017/4/11 0:00:00

Tandem expression and activity determination of antibacterial peptide Spinosan-C from Paa spinosa
Yue Liu,Zhonggen Zhan,Bing Zhu,Rongquan Zheng,Hongyi Cheng and Zuoming Nie.Tandem expression and activity determination of antibacterial peptide Spinosan-C from Paa spinosa[J].Chinese Journal of Biotechnology,2018,34(1):132-139.
Authors:Yue Liu  Zhonggen Zhan  Bing Zhu  Rongquan Zheng  Hongyi Cheng and Zuoming Nie
Institution:1 Zhejiang Institute of Economics and Trade, Hangzhou 310018, Zhejiang, China,1 Zhejiang Institute of Economics and Trade, Hangzhou 310018, Zhejiang, China,2 College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, Zhejiang, China,3 Institute of Ecology, Zhejiang Normal University, Jinhua 321004, Zhejiang, China,3 Institute of Ecology, Zhejiang Normal University, Jinhua 321004, Zhejiang, China and 2 College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, Zhejiang, China
Abstract:Antibacterial peptide can be easily degraded by protease and has the lethal effect on the host Escherichia coli. In order to solve these problems and further improve the expression ability of the Escherichia coli system, the antimicrobial peptide Spinosan-C of Paa spinosa was studied. First, the codon of Spinosan-C was optimized according to E. coli codon usage frequency. Then, the 8 multimeric Spinosan-C gene (8×Spinosan-C) was synthesized and cloned into prokaryotic expression vector pET-28a. The fusion antimicrobial peptide 8×Spinosan-C was further highly expressed in Escherichia coli strain Rosetta. The recombinant 8×Spinosan-C protein was then purified and cleaved specially by formic acid to generate the Spinosan-C monomer. Antibacterial test in vitro suggested that the cleaved Spinosan-C monomer had antibacterial bioactivity against the test bacteria. This study provides a technical reference for the largescale preparation of frog antimicrobial peptides.
Keywords:Paa spinosa antimicrobial peptide  Spinosan-C  tandem expression  specific cleaving  antibacterial activity
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