Immune Response and Antioxidant Activities of Bacteria-stimulated Prx4 in Procambarus clarkii

  Objective  Immune response of peroxiredoxin 4 gene of Procambarus clarki (Pc-prx4) after being stimulated by bacterial infection was studied.

  Methods  Pc-prx4 were cloned before and after an artificial inoculation of Staphylococcus aureus or Edwardsiella ictaluri on the crayfish. Bioinformatic analysis was conducted with the aid of relevant online websites and software including Expasy, translate tool, SMART, Expasy ProtParam tool, BLASTx, and MEGA-X. Expressions of Pc-prx4 from the tissues of normal and pathogenically inoculated crayfish were detected by qRT-PCR. Antioxidant function of the constructed recombinant protein was determined.

  Results   Pc-prx4 had an open reading frame of 744 bp encoding 247 amino acids with an alkyl hydroperoxide reductase (AhpC) structural domain, a sulfhydryl-specific antioxidant structural domain, and a C-terminal structural domain of 1-Cys prx peroxisome iron oxidase. The predicted protein molecular formula was C₁₂₄₉H₁₉₃₄N₃₃₀O₃₅₇S₁₀ with a molecular weight of 27.61 kDa and a pI of 5.88. Its signal peptide region was located at the N-terminal end, and the 1-Cys peroxiredoxin domain at the C-terminal end. The gene expressed in all sampled tissues—most highly in the hemolymph and high in the hemocytes, hepatopancreas, gills, and intestines of the crayfish inoculated by S. aureus or E. catarrhalis. In the plasmid DNA protection assay, rPc-PRX4 displayed varying degrees of antioxidant activity, especially in high concentrations.

  Conclusion  A kind of 1-Cys prx, Pc-prx4 was highly expressed in the hemolymph of P. clarkii stimulated by S. aureus or E. catarrhalis. The protein exhibited a concentration-dependent activity associated with the antioxidant homeostasis of the crayfish.