Full-length cDNA cloning, molecular characterization and differential expression analysis of peroxiredoxin 6 from Ovis aries

Vet Immunol Immunopathol. 2015 Apr 15;164(3-4):208-19. doi: 10.1016/j.vetimm.2015.01.006. Epub 2015 Jan 28.

Abstract

Peroxiredoxin 6 (Prdx6), an important antioxidant enzyme that can eliminate reactive oxygen species (ROS) to maintain homeostasis, is a bifunctional protein that possesses the activities of both glutathione peroxidase and phospholipase A2. In this study, a novel full-length Prdx6 cDNA (OaPrdx6) was cloned from Sheep (Ovis aries) using rapid amplification of cDNA ends (RACE). The full-length cDNA of OaPrdx6 was 1753bp containing a 5'-untranslated region (UTR) of 93bp, a 3'-UTR of 985bp with a poly(A) tail, and an open reading frame (ORF) of 675bp encoding a protein of 224 amino acid residues with a predicted molecular weight of 25.07kDa. The recombinant protein OaPrdx6 was expressed and purified, and its DNA protection activity was identified. In order to analyze the Prdx6 protein expression in tissues from O. aries, monoclonal antibodies against OaPrdx6 were prepared. Western blotting results indicated that OaPrdx6 protein could be detected in heart, liver, spleen, lung, kidney, stomach, intestine, muscle, lymph node and white blood cells, and the highest expression was found in lung while the lowest expression in muscle. Compared to the normal sheep group, the mRNA transcription level of Prdx6 in buffy coat was up-regulated in the group infected with a virulent field strain of Brucella melitensis, and down-regulated in the group inoculated with a vaccine strain S2 of brucellosis. The results indicated that Prdx6 was likely to be involved in the host immune responses against Brucella infection, and probably regarded as a molecular biomarker for distinguishing between animals infected with virulent Brucella infection and those inoculated with vaccine against brucellosis.

Keywords: Brucellosis; Expression analysis; Monoclonal antibodies; Ovis aries; Peroxiredoxin 6.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal / immunology
  • Base Sequence
  • Brucella melitensis / immunology
  • Brucellosis / immunology
  • Brucellosis / veterinary
  • Cloning, Molecular
  • DNA, Complementary / chemistry
  • DNA, Complementary / genetics*
  • Molecular Sequence Data
  • Peroxiredoxin VI / genetics*
  • Peroxiredoxin VI / immunology
  • Recombinant Proteins / immunology
  • Sheep

Substances

  • Antibodies, Monoclonal
  • DNA, Complementary
  • Recombinant Proteins
  • Peroxiredoxin VI