Recombinant expression and functional analysis of antimicrobial Siganus oramin L-amino acid oxidase using the Bac-to-Bac baculovirus expression system

摘要

Siganus oramin L-amino acid oxidase (SR-LAAO), isolated from the serum of Siganus oramin, is an innate immune protein with significant antibacterial activity. The aim of this study was to express SR-LAAO in insect cells using a baculovirus expression system and evaluate the function of the recombinant SR-LAAO. To this end, an optimized sequence of the SR-LAAO gene was designed and synthesized, based on the codon bias of insect cells. Bacmid shuttle vectors and recombinant baculovirus were successfully constructed, and the recombinant baculovirus was transfected into Sf9 insect cells. The antibacterial activity and enzymatic characteristics of the recombinant SR-LAAO were investigated. The results showed that the pFastBac-optiSR-LAAO shuttle vectors and Bacmid-optiSR-LAAO were correctly constructed. The Sf9 insect cells exhibited significant cytopathic effects following infection with Bacmid-optiSR-LAAO and Bacmid; the specific PCR analysis proved that the recombinant baculovirus was successfully constructed. The immunofluorescence assay revealed that the recombinant baculovirus rSR-LAAO was abundantly expressed in infected Sf9 insect cells; the results of SDS/PAGE and Western blot analyses showed that a specific band appeared at about 60 kDa. Moreover, the crude rSR-LAAO enzyme displayed strong antibacterial activity against aquatic pathogens, particularly Streptococcus agalactiae and Streptococcus iniae. In addition, the results of catalase interference test implied that the antibacterial activity of rSR-LAAO was directly associated with (H2O2 production). The results of the rSR-LAAO enzymatic characteristics test indicated that the Km value with L-Lysine as a substrate was 16.61 mM when the temperature was under 37 degrees C, and the optimum pH was 7. The antibacterial activity of rSR-LAAO could be completely inhibited by 10 mg/mL of pepsin, trypsin, and proteinase K compared with both methanol and acetone. Adding an equal volume of ethanol had a minimal impact on the antibacterial activity of rSR-LAAO. The crude enzyme could maintain a high level of antibacterial activity against both Gram-positive and -negative bacteria from 4 degrees C to 30 degrees C. In the present study, SR-LAAO was successfully expressed in Sf9 cells using the baculovirus expression system, and provides basic references for further research into the role of LAAO in marine animals and the development of new antimicrobial drugs.