Molecular cloning, expression and antioxidant activity of a peroxiredoxin 2 homologue from Lampetra japonica.
Peroxiredoxin (Prx) is a cellular antioxidant protein family that plays important roles in oxidative stress and immune cytotoxicity. In this study, we cloned a homologue of the Prx2 from the buccal gland of Lampetra japonica (L. japonica). L. japonica Prx2 (Lj-Prx2) contained two highly conserved motifs and shared more than 70% identity with the homologs from other vertebrate species. Phylogenetic analysis revealed that Lj-Prx2 is closely related to other available teleost Prx2. The real-time PCR results demonstrated that the Prx2 gene was widely expressed in adult lamprey. In addition, the expression of Prx2 gene was particularly up-regulated in red blood cells (RBCs) after the experimental animals were challenged with lipopolysaccharide (LPS) in vivo. Lj-Prx2 gene was subcloned into the pET23b vector and expressed in Escherichia coli BL21 (DE3). The recombinant L. japonica Prx2 (rLj-Prx2) was purified by using His Bind affinity chromatography. Polyclonal antibody to rLj-Prx2 was generated in New Zealand Rabbit. Western blot analysis showed that the Lj-Prx2 is present in the buccal gland secretion, suggesting the secretory feature of it. The function assays revealed that rLj-Prx2 has the capability to reduce the H2O2 when dithiothreitol (DTT) is used as a reducing equivalent and to protect DNA from oxidative damage. These findings suggested that Lj-Prx2 probably plays an essential role in antioxidant defense in RBCs to keep lamprey alive.
Sun
Jing
J
Institute of Marine Genomics & Proteomics, Liaoning Normal University, 850 Huanghe Road, Dalian 116029, China.
Liu
Xin
X
Li
Qingwei
Q
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D007798:Lampreys / Q000145:classification / Q000235:genetics* / Q000276:immunology*; D008969:Molecular Sequence Data; D054464:Peroxiredoxins / Q000235:genetics* / Q000276:immunology*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D011994:Recombinant Proteins; D054464:Peroxiredoxins