厚壳贻贝Mytilin-1成熟肽在毕赤酵母中的重组表达及其抑菌活性

doi:10.13560/j.cnki.biotech.bull.1985.2019-0104

摘要/Abstract

摘要： 贻贝素（Mytilins）是一类主要在贝类血细胞中表达的阳离子小分子抗菌肽,包括多种同工型,它们在免疫系统中发挥了重要作用,Mytilin-1是其中的一种同工型。厚壳贻贝（Mytilus coruscus）Mytilin-1的一级结构由信号肽、成熟肽和C端的延伸肽组成;其成熟肽决定了Mytilin-1的生物学活性,它由34个氨基酸残基组成,其中8个保守的半胱氨酸残基在空间上能形成4对二硫键,对Mytilin-1的稳定性和活性起了关键作用。以厚壳贻贝的Mytilin-1成熟肽为重组DNA表达的目的蛋白,根据毕赤酵母（Pichia pastoris）的密码子偏爱性对编码该成熟肽的密码子进行优化,合成的目的基因“mMy1”与表达载体pPICZαA连接后电转至毕赤酵母X-33,通过高浓度博来霉素筛选高拷贝酵母转化子,获得的阳性转化子在29℃、250 r/min、pH6.0的条件下,使用1%甲醇诱导表达96 h;培养液上清经Tricine-SDS-PAGE和Western blot分析证明：重组mMy1在毕赤酵母X-33中得到成功表达,其分子量为4.8 kD。抑菌试验表明,重组mMy1对革兰氏阳性的金黄色葡萄球菌（Staphylococcus aureus）和枯草芽孢杆菌（Bacillus subtilis）以及革兰氏阴性的大肠杆菌（Escherchia coli）具有抑菌活性。

关键词: 厚壳贻贝, Mytilin-1成熟肽, 毕赤酵母, 重组表达, 抑菌活性

Abstract: Mytilins are small cationic antibacterial peptides expressed predominantly in the haemocytes of shellfish,including multiple isoforms,and they play an important role in the innate immune system of shellfish. Mytilin-1 is one of the isoforms. The primary structure of Mytilus coruscus mytilin-1 is composed of signal peptide,mature peptide and C-terminal extension peptide. The mytilin-1’s mature peptide consisting of 34 amino acid residues determines its biological activity;8 conserved cysteine residues in the mature peptide may form 4 pairs of disulfide bond to stabilize the structure of mytilin-1 mature peptide. In the present study,having mytilin-1 mature peptide from M. coruscus as the recombinant DNA-expressed protein,a codon-optimized sequence（named as “mMy1”）corresponding to the cDNA encoding mytilin-1 mature peptide was synthesized based on the preferential codon usage of Pichia pastoris,and ligated to the expression vector pPICZαA and electronically transformed into competent P. pastoris X-33. The positive transformants containing multi-copy gene insertions were screened using high-concentration zeocin. The recombinant mMy1 was induced for 96 h with 1.0% methanol at 29℃,250 r/min and pH6.0. Tricine-SDS-PAGE and Western blot analysis demonstrated that the recombinant mMy1 with a molecular mass of 4.8 kD was expressed successfully in P. pastoris X-33. Bacteriostatic test indicated that the culture medium supernatant containing recombinant mMy1 showed antibacterial activity against Gram-positive（i.e.,Staphylococcus aureus and Bacillus subtilis）and Gram-negative（i.e.,Escherchia coli）bacteria.

Key words: Mytilus coruscus, mytilin-1 mature peptide, Pichia pastoris, recombinant expression, antibacterialactivity

张亚莉, 陶妍, 谢晶, 钱韻芳. 厚壳贻贝Mytilin-1成熟肽在毕赤酵母中的重组表达及其抑菌活性[J]. 生物技术通报, 2019, 35(7): 54-60.

ZHANG Ya-li, TAO Yan, XIE Jing, QIAN Yun-fang. Recombinant Expression of Mytilus coruscus Mytilin-1 Mature Peptide in Pichia pastoris and Its Antibacterial Activity[J]. Biotechnology Bulletin, 2019, 35(7): 54-60.

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