Main content area

Molecular cloning, expression and antioxidative activity of 2-cys-peroxiredoxin from freshwater mussel Cristaria plicata

Wang, Xiaobo, Hu, Baoqing, Wen, Chungen, Zhang, Ming, Jian, Shaoqing, Yang, Gang
Fish & shellfish immunology 2017 v.66 pp. 254-263
Aeromonas hydrophila, Escherichia coli, antioxidant activity, complementary DNA, cysteine, freshwater mussels, gene expression, gene expression regulation, gills, hemocytes, hepatopancreas, hydrogen peroxide, hydroperoxides, messenger RNA, molecular cloning, open reading frames, oxidative stress, peptidoglycans, peroxiredoxin, plasmids, quantitative polymerase chain reaction, recombinant proteins, sequence homology
Peroxiredoxins (Prxs) play an important role against various oxidative stresses by catalyzing the reduction of hydrogen peroxide (H2O2) and organic hydroperoxides to less harmful form. A 2-cys peroxiredoxin, designated as CpPrx, was cloned from hemocytes of freshwater mussel Cristaria plicata. The full length cDNA of CpPrx is 1247 bp, which includes an open reading frame (ORF) of 591bp, encoding 196 amino acids. CpPrx possesses two conserved cysteine residues (Cys49, Cys170). The deduced amino acid sequence of CpPrx showed a high level (67–74%) of sequence similarity to 2-Cys Prxs from other species. The results of real-time quantitative PCR revealed that CpPrx mRNA was constitutively expressed in tissues, and the highest expression levels were in hepatopancreas and gills. After peptidoglycan (PGN) and Aeromonas hydrophila challenge, the expression levels of CpPrx mRNA were up-regulated in hemocytes and hepatopancreas. The cDNA of CpPrx was cloned into the plasmid pET-32, and the recombinant protein was expressed in Escherichia coli BL21(DE3). Comparison with DE3-pET-32 and DE3 strain, the cells of DE3-pET-32-CpPrx exhibited resistance to the concentration of 0.4, 0.8 and 1.2 mmoL/L H2O2 in vivo.