Molecular cloning,expression and antioxidant activity of a peroxiredoxin 2 homologue from Lampetra japonica |
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| Authors: | Jing Sun Xin Liu Qingwei Li |
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| Affiliation: | 1. Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China;2. University of Chinese Academy of Sciences, Beijing 100049, PR China;1. Christchurch Heart Institute, University of Otago, Christchurch, New Zealand;2. Cardiovascular Research Institute, National University of Singapore, Singapore;1. Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China;2. University of Chinese Academy of Sciences, Beijing 100049, China;3. Department of Chemistry, Texas A&M University, College Station, Texas 77843;4. Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843;3. Department of Surgery, Public Health, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425;4. Department of Microbiology and Immunology, Public Health, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425;6. Department of Biochemistry and Molecular Biology, Public Health, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425;8. Department of Pharmaceutical and Biomedical Sciences, Public Health, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425;9. Public Health, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425;5. Department of Surgery, Loyola University, Maywood, Illinois 60153;12. Department of Surgery, University of Connecticut Health Center, Farmington, Connecticut 06030 |
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| Abstract: | 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. |
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