1. Construction and activity analyses of single functional mouse peroxiredoxin 6 (Prdx6)
- Author
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Hong-Lin Ren, Zhang Shijun, Yan-Song Li, Yu Zhou, Dan-Di Ju, Pan Hu, Zeng-Shan Liu, Bing Su, Lu-Lu Wang, Shi-Ying Lu, Bao-Quan Fu, and Fei-Fei Zhai
- Subjects
040301 veterinary sciences ,Veterinary medicine ,Mutant ,law.invention ,0403 veterinary science ,03 medical and health sciences ,Plasmid ,law ,SF600-1100 ,phospholipase a2 ,Overlap extension polymerase chain reaction ,glutathione peroxidase ,mouse ,peroxiredoxin 6 ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,General Veterinary ,biology ,Glutathione peroxidase ,04 agricultural and veterinary sciences ,Transfection ,Molecular biology ,Enzyme assay ,Open reading frame ,chemistry ,Recombinant DNA ,biology.protein ,soe-pcr ,Research Article - Abstract
Introduction: Peroxiredoxin 6 (Prdx6) is a bifunctional protein with glutathione peroxidase activity and phospholipase A2 activity. Previous studies have shown a significant positive correlation between the intracellular survival ability of Brucella and Prdx6. Here, the Prdx6 enzyme with a single activity was constructed to facilitate study of the relationship between the single function of Prdx6 and Brucella infection. Material and Methods: The target open reading frame (ORF) DNAs of Prdx6 with a single active centre were prepared using gene splicing by overlap extension PCR (SOE-PCR), and the recombinant eukaryotic expression plasmids inserted by Prdx6 with the single activity centre were constructed and transfected into murine Raw264.7 macrophages. The glutathione peroxidase activity and phospholipase A2 activity of the constructed Prdx6 were examined. Results: The core centres (Ser32 and Cys47) of Prdx6 were successfully mutated by changing the 94th nucleotide from T to G and the 140th nucleotide from G to C in the two enzyme activity cores, respectively. The constructed recombinant plasmids of Prdx6 with the single active centre were transfected into murine macrophages showing the expected single functional enzyme activity, which MJ33 or mercaptosuccinate inhibitors were able to inhibit. Conclusion: The constructed mutants of Prdx6 with the single activity cores will be a benefit to further study of the biological function of Prdx6 with different enzyme activity.
- Published
- 2019