农业生物技术学报2012，Vol.20Issue(2)：196-202,7.DOI:10.3969/j.issn.1674-7968.2012.02.012
在毕赤酵母中利用口蹄疫病毒(FMDV)2A实现dTomato和串联MagaininⅡ基因的共表达
Coexpression of dTomato and Muti-copy Magainin Ⅱ Linked by the Foot-and mouth disease virus (FMDV) 2A Region in a Single Open Reading Frame in Pichia pastoris
摘要
Abstract
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 GS\l5/pPlC9K-dTomato-2A-3M. Observed under a fluorescence microscope, supernatant of GSl\5/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.关键词
抗菌肽/MagaininⅡ/口蹄疫病毒(FMDV) 2A/共表达Key words
Antibacterial peptide, Magainin Ⅱ, Foot-and mouth disease virus(FMDV) 2A, Coexpression引用本文复制引用
卢艳敏,崔海信,王承民,何宏轩..在毕赤酵母中利用口蹄疫病毒(FMDV)2A实现dTomato和串联MagaininⅡ基因的共表达[J].农业生物技术学报,2012,20(2):196-202,7.基金项目
中国科学院绿色农业技术集成与发展中心创新方向项目(No.Y029891134)和战略生物资源科技支撑体系项目(No.ZBZX-12011) （No.Y029891134）
