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当前位置: 首页 > 文章 > 光滑鳖甲抗菌肽的原核表达条件优化及其抗菌活性 昆虫学报 2016,59 (1) 8-15
Position: Home > Articles > Optimization of prokaryotic expression conditions and the antibacterial activity of a novel antimicrobial peptide from Anatolica polita borealis(Coleoptera: Tenebrionidae) Acta Entomologica Sinica 2016,59 (1) 8-15

光滑鳖甲抗菌肽的原核表达条件优化及其抗菌活性

作  者：
海力且木·艾力;徐鑫;刘忠渊
单  位：
新疆大学生命科学与技术学院新疆生物资源基因工程重点实验室
关键词：
光滑鳖甲;抗菌肽;原核表达;蛋白纯化;抗菌活性
摘  要：
【目的】本研究旨在探索光滑鳖甲Anatolica polita borealis抗菌肽Ap AMP1015的最佳原核表达条件及其抗菌活性。【方法】利用生物信息学方法对得到的Ap AMP1015基因序列和蛋白结构进行分析,运用原核表达技术表达Trx A-Ap AMP1015融合蛋白,通过Western blot方法鉴定蛋白,并利用亲和层析的方法获得纯化的Trx A-Ap AMP1015融合蛋白,抑菌圈实验验证蛋白抗菌活性。【结果】克隆得到光滑鳖甲抗菌肽基因Ap AMP1015,其开放阅读框长387 bp,编码128个氨基酸,其中包含由19个氨基酸组成的信号肽和75个氨基酸组成的成熟肽。NCBI数据库同源序列比对结果显示该蛋白属Coleoptericin抗菌肽家族。确定了蛋白表达的最佳条件:0.1 mmol/L IPTG 150 r/min 25℃诱导4 h。肠激酶切割后的Ap AMP1015能够有效抑制大肠杆菌Escherichia coli的生长。【结论】克隆得到光滑鳖甲抗菌肽基因Ap AMP1015,获得了其编码蛋白的最优表达条件,研究发现Ap AMP1015能够有效抑制大肠杆菌的生长。本研究为光滑鳖甲抗菌肽Ap AMP1015的应用和进一步研究奠定了基础。
译  名：
Optimization of prokaryotic expression conditions and the antibacterial activity of a novel antimicrobial peptide from Anatolica polita borealis(Coleoptera: Tenebrionidae)
作  者：
HAILIQIEMU AiLi;XU Xin;LIU Zhong-Yuan;Xinjiang Key Laboratory of Biological Resources and Genetic Engineering,College of Life Science and Technology,Xinjiang University;
关键词：
Anatolica polita;;antimicrobial peptide;;prokaryotic expression;;protein purification;;antibacterial activity
摘  要：
【Aim】To explore the optimal prokaryotic expression conditions and study the antimicrobial activity of a novel glycine-rich antimicrobial peptide Ap AMP1015 from Anatolica polita borealis.【Methods】The c DNA and deduced amino acid sequences of Ap AMP1015 were analyzed by bioinformatics methods. Prokaryotic expression technology was used to express fusion protein Trx A-Ap AMP1015. The fusion protein Trx A-Ap AMP1015 was verified by Western blot,and purified with affinity chromatography.Inhibition zone experiment was made to assay the antibacterial activity of Ap AMP1015. 【Results】An antimicrobial peptide gene Ap AMP1015 was cloned from A. polita borealis. Ap AMP1015 has the opening reading frame of 387 bp and encodes a 128-amino-acid peptide consisting of a signal peptide of 19 amino acids and a mature peptide of 75 amino acids. Homology analysis showed that Ap AMP1015 is a member of coleoptericins. The prokaryotic expression vector p ET-32a-Ap AMP1015 was successfully constructed and the recombinant protein expressed in Escherichia coli BL21( DE3) was verified by Western blot. The0. 1 mmol / L IPTG induced a higher level of expression of recombinant protein in the supernatant in the culture condition of 25℃ and 150 r / min. The inhibition zone experiment showed that Ap AMP1015 exhibited the antibacterial activity against E. coli. 【Conclusion】The full-length c DNA of Ap AMP1015 was cloned from A. polita borealis. The optimal prokaryotic expression conditions were determined.Ap AMP1015 can inhibit the growth of E. coli. This study lays the foundation for promoting the application and further study of Ap AMP1015 from A. polita borealis.

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