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

Affiliation

Institute of Agricultural Resources and Environment, Jiangsu Academy of Agricultural Sciences, Nanjing, China; Key Laboratory of Agro-Environment in Downstream of Yangtze Plain, Ministry of Agriculture, Nanjing, China. Electronic address: [Email]

Abstract

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.

Keywords

Bacterial agglutination assay,Copper/zinc superoxide dismutase (Cu/ZnSOD),Immune gene expression,Macrobrachium rosenbergii,Saccharomyces cerevisiae,Vibrio parahaemolyticus,