Molecular characterization of Peroxiredoxin-6 (Prx6) from sevenband grouper (Hyporthodus septemfasciatus) and evaluation of its antiviral effect against NNV

다국어 초록 (Multilingual Abstract)

The seven-band grouper (Hyporthodus septemfasciatus) is a high-value aquaculture species, but viral nervous necrosis (VNN) frequently occurs during the seedling stage under high-temperature conditions. As NNV-induced oxidative stress activates host antioxidant enzymes, the role of peroxiredoxin 6 (Prx6) in this response warrant’s investigation. Prx6 is a well-characterized antioxidant enzyme that maintains cellular redox homeostasis and participates in H₂O₂-mediated signaling pathways. In this study, we identified the full-length cDNA of HsPrx6, which contains a 666 bp open reading frame (ORF) encoding a 221-amino-acid protein with a predicted molecular mass of 24.27 kDa. Structural modeling confirmed the conserved 1-Cys Prx motif (⁴⁴PVCTTE⁴⁹) and catalytic triad (T⁴³, C⁴⁶, R¹³⁰). Quantitative real-time PCR analysis revealed that HsPrx6 was ubiquitously expressed across tissues, with the highest levels in the spleen, followed by gill, liver, brain, heart, and kidney. Following NNV infection, HsPrx6 expression was significantly up-regulated in all examined tissues. For recombinant expression, HsPrx6 was cloned into the pColdⅠ vector and expressed in E. coli BL21(DE3). The purified recombinant protein exhibited strong antioxidant activity, as increasing protein concentrations enhanced H₂O₂ clearance and reduced DNA damage in the metal-catalyzed oxidation (MCO) assay. In SSN-1 cells transfected with pcDNA3.1(+)_HsPrx6, NNV RNA2 copy number was significantly decreased. Mitochondrial membrane potential (MMP) was preserved as confirmed by JC-1 staining, and WST-1 assays showed improved cell viability in the HsPrx6 overexpession cells. Moreover, the expression of immune- and stress-related genes such as HSP70, NF-κB p65, IκBα, TNFα, IL-8, IL-6, Viperin, IRF7 was elevated. These findings suggest that Prx6 modulates ROS generated during NNV infection. By doing so, it activates intracellular redox signaling pathways—including NF-κB-mediated inflammatory responses—which contribute to enhanced antiviral defense and cellular protection.

목차 (Table of Contents)

• Ⅰ. Introduction 1
• Ⅱ. Materials & Methods 4
• 1. Fish and cell culture 4
• 2. Total RNA extraction and cDNA synthesis 4
• 3. Virus propagation and titer determination 5
• 4. In vivo HsPrx6 expression analysis 6
• 5. Identification and bioinformatics analysis of HsPrx6 8
• 6. Cloning of HsPrx6 cDNA 9
• 7. Construction of HsPrx6 expression vector 10
• 8. Expression and purification of recombinant HsPrx6 12
• 9. SDS-PAGE 13
• 10. Western blot analysis of recombinant HsPrx6 13
• 11. Antioxidant assay of rHsPrx61 14
• 12. Expression analysis of HsPrx6 in SSN-1 cells after transfection 15
• 13. Preparation of standard curves for absolute quantification of NNV RNA 16
• 14. HsPrx6 overexpression and NNV infection in SSN-1 Cells 18
• 15. qPCR analysis of NNV replication and immune-related gene expression 18
• 16. Determination of viral titer in culture supernatants 19
• 17. Measurement of mitochondrial membrane potential (∆ΨM) 19
• 18. Cell viability 20
• 19. Statistical analysis 20
• Ⅲ. Results 21
• 1. The identification and bioinformatics analysis of HsPrx6 21
• 2. In vivo HsPrx6 expression analysis 26
• 3. Over-expression and western blotting assay of rHsPrx6 28
• 4. Antioxidant activity of rHsPrx6 31
• 5. Expression pattern of HsPrx6 during transfection in SSN-1 cell 33
• 6. Antiviral effects of HsPrx6 overexpression in NNV-infected SSN-1 cells 35
• 7. Effects of HsPrx6 overexpression on immune-related gene expression in NNV-infected SSN-1 cells 37
• 8. Protective effect of HsPrx6 overexpression on mitochondrial function in NNV-infected SSN-1 cells 39
• 9. Cytoprotective effect of HsPrx6 overexpression in NNV-infected SSN-1 cells 41
• Ⅳ. Discussion 43
• Ⅴ. 국문 초록 50
• Ⅵ. References 51