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  

Recombinant Expression of Mytilus coruscus Mytilin-1 Mature Peptide in Pichia pastoris and Its Antibacterial Activity

作　　者：张亚莉 陶妍 谢晶 钱韻芳 ZHANG Ya-li;TAO Yan;XIE Jing;QIAN Yun-fang(Shanghai Engineering Research Center of Aquatic-Product Processing&Preservation,College of Food Science and Technology,Shanghai Ocean University,Shanghai 201306)

机构地区：[1]上海海洋大学食品学院上海水产品加工及贮藏工程技术研究中心

出　　处：《生物技术通报》2019年第7期54-60,共7页Biotechnology Bulletin

基　　金：国家“十三五”重点研发计划(2016YFD0400106)

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。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.

关 键 词：厚壳贻贝 Mytilin-1成熟肽 毕赤酵母 重组表达 抑菌活性 

分 类 号：TS2[轻工技术与工程—食品科学与工程]