Macrobrachium rosenbergii Cu/Zn superoxide dismutase (Cu/Zn SOD) expressed in Saccharomyces cerevisiae and evaluation of the immune function to Vibrio parahaemolyticus

• Published in: Fish & shellfish immunology 2019-07, Vol.90, p.363-375
• Main Authors: Du, Jie, Zhu, Huanxi, Ye, Minshuo, Ma, Yan
• Format: Article
• Language: English
• Subjects: Bacterial agglutination assay; Copper/zinc superoxide dismutase (Cu/ZnSOD); Immune gene expression; Macrobrachium rosenbergii; Saccharomyces cerevisiae; Vibrio parahaemolyticus
• ISSN: 1050-4648; 1095-9947
• DOI: 10.1016/j.fsi.2019.04.016

Superoxide dismutases (SODs) are important antioxidant enzymes that occur in virtually all oxygen-respiring organisms, and copper/zinc SOD (Cu/ZnSOD) is one of the most important SODs. In the present study, Macrobrachium rosenbergii Cu/Zn-SOD was expressed in a yeast eukaryotic system. The open reading frame (ORF) of MrCu/ZnSOD was cloned into the plasmid vector pHAC181, and the recombinant plasmid was integrated into the downstream region of the GAL1 promoter in Saccharomyces cerevisiae strain GAL1-ScRCH1 via homologous recombination. The resulting recombinant MrCu/ZnSOD consisted of a 3 × HA-tag at its C-terminal. Via western blot, the molecular weight of the recombinant MrCu/ZnSOD was estimated at about 30 kDa. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of this recombinant MrCu/ZnSOD ranged from 0.556 to 0.840 μM, and from 0.967 to 2.015 μM, respectively. The recombinant MrCu/ZnSOD protein was able to agglutinate four Gram-negative bacterial strains, as well as two of three Gram-positive strains (except Staphylococcus aureus). This demonstrated that the recombinant protein possessed some antimicrobial activity against certain Gram-positive and Gram-negative bacteria. M. rosenbergii were fed with the recombinant yeast strain MrCu/ZnSOD for 4 weeks and then challenged with the most common crustacean pathogen, Vibrio parahaemolyticus. This group of prawns presented lower mortality, higher enzymatic activity, and higher expression of the mRNA of immune-related genes than that in the control groups. Taken together, these results suggest that MrCu/ZnSOD is an antioxidant enzyme and antimicrobial peptide involved in the crustacean innate immune system and offers protection to the host against pathogenic bacteria. •MrCu/ZnSOD was expressed in S. cerevisiae and its MW was about 30 kDa.•MIC and MBC of MrLGBP were ranged from 0.556 to 0.840 μM and 0.967–2.015 μM.•MrCu/ZnSOD can agglutinate four Gram-positive and two negative bacteria species.•MrCu/ZnSOD enhanced the immunity of M. rosenbergii against V. parahaemolyticus.