|
|
|
| Coexpression of dTomato and Muti-copy MagaininⅡLinked by the Foot-and mouth disease virus (FMDV) 2A Region in a Single Open Reading Frame in Pichia pastoris |
| 2, 2, 2 |
|
|
|
|
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 MagaininⅡgenes 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 (AOX1) 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 GS115/pPIC9K-dTomato-2A-3M. Observed under a fluorescence microscope, supernatant of GS115/pPIC9K-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 MagaininⅡ) 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.
|
|
Received: 14 July 2011
|
|
|
|
|
|
|
