抗菌肽(antibacterial peptides)是生物体内经诱导产生的一种具有生物活性的多肽,是天然免疫的重要组成部分.为了提高抗菌肽的表达量以及很方便地检测抗菌肽的表达情况,本研究利用口蹄疫病毒(Foot-and-mouth disease virus,FMDV) 2A将荧光蛋白dTomato基因和3个串联的MagaininⅡ基因融合到一个开放阅读框中(open reading frame,ORE),融合的基因被置于pPIC9K载体醇氧化酶基因(AOX1)启动子的下游,构建分泌型重组酵母表达载体pPIC9K-dTomato -2A-3M,将线性化的重组酵母表达载体通过电击法转入毕赤酵母(Pichia pastoris )GS115中,经G418筛选和PCR鉴定得到阳性转化子,然后将其转至摇瓶,在30℃、0.5％甲醇的条件下进行诱导表达,连续诱导3d.SDS-PAGE电泳图谱显示,在31 kD(dTomato)和9.5kD(3M)处有蛋白条带出现,在荧光显微镜下观察酵母表达上清发出红色荧光,对大肠杆菌(Escherichia coli)和金黄色葡萄球菌(Staphylococcus aureus)抑菌试验结果表明,重组抗菌肽串联的MagaininⅡ具有明显的抑菌效果.结果表明,由2A连接的融合基因(荧光蛋白dTomato和串联的MagaininⅡ)在毕赤酵母中成功的进行了表达,FMDV 2A在其C端剪切多聚蛋白,得到dTomato和串联的MagaininⅡ两个独立而有活性的蛋白,为小分子抗菌肽的表达提供一个高效的检测方法.%Antimicrobial peptideis is a kind of bioactive polypeptides induced by pathogens in organisms, which is an important component of innate immunity. In order to improve the expression of antimicrobial peptide and easily detect the expression of antimicrobial peptide. The red fluorescent protein dTomato and muti-copy Magainin II genes were fused into a single open reading frame (ORF) with a copy of the Foot-and mouth disease virus (FMDV) 2A region placed between the two genes. The fused genes were placed under the control of the alcohol oxidase (A 0X1) gene promoter in pPIC9K vector to construct the recombinant expression vector T-dTomato-2A-3M. The linearized plasmid T-dTomato-2A-3M was transformed into Pichia pastoris GS115 strain by electroporation to construct the recombinant expression yeast strain. Through G418 screening and PCR identification, the positive clones were transferred into shake flasks at 30℃ with 0.5% methanol to induct for 3 d. SDS-PAGE analysis showed that the recombinant had expressed a 31 and a 9.5 kD proteins in supernatant of GSl5/pPlC9K-dTomato-2A-3M. Observed under a fluorescence microscope, supernatant of GSl5/pPlC9K-dTomato-2A-3M appeared red fluorescent. The activity assay demonstrated that the recombinant antimicrobial peptide had antibacterial activities against Escherichia coli and Staphylococcus aureus. The results show that the fused genes (red fluorescent protein dTomato and muti-copy Magainin Ⅱ) linked by 2A region of FMDV are expressed in P. Pastoris successfully and the polyprotein is "cleaved" to each functional protein, which provides an effective method to detect the expression of antimicrobial peptided.
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