Molecular Characterization and Functional Analysis of Tumor Necrosis Factor from Sevenband Grouper (Hyporthodus septemfasciatus) in Response to Nervous Necrosis Virus Infection
- Abstract
- Tumor necrosis factor (TNF-α) is a key pro-inflammatory cytokine involved in im-mune regulation. In this study, the TNF-α gene from sevenband grouper (HsTNF-α) was cloned, and recombinant HsTNF-α protein (rHsTNF-α) was expressed in Escherichia coli (E. coli). Its antiviral effects against nervous necrosis virus (NNV) were assessed in vitro. The open reading frame (ORF) of HsTNF- α was 762 bp, en-coding a 253 aa contained the TNF homology domain and transmembrane domain. The rHsTNF-α was expressed in E. coli, purified via Ni-NTA nanobead, and con-firmed to be approximately 20.5 kDa by SDS-PAGE and western blotting. The func-tional activity of rHsTNF-α was assessed by treating SSN-1 cells with various con-centrations of protein, followed by NNV infection. Viral RNA2 copy number de-creased at 24 hours post-infection (hpi) and remained suppressed through 72 hpi.
The expression levels of TNF-related genes were examined. Caspase-3 showed a significant increase in expression, whereas caspase-8 and Fas-associated with death domain(FADD) exhibited low expression levels, suggesting possible post-transcriptional regulation. Furthermore, increased expression of IκBα and TNFα was observed significantly up-regulation. These results indicate activation of both the apoptotic and NF-kB signaling pathways at the mRNA level.
- Issued Date
- 2025
- Awarded Date
- 2025-08
- Type
- Dissertation
- Publisher
- 국립부경대학교 대학원
- Alternative Author(s)
- 서정민
- Affiliation
- 국립부경대학교 대학원
- Department
- 대학원 미생물학과
- Advisor
- Jongoh Kim
- Table Of Contents
- 1. Introduction 1
2. Materials & Methods 5
2.1. Fish 5
2.2. Cell culture 6
2.3. Virus propagation and titer determination 7
2.4. Identification and cloning of HsTNF-α sequence 8
2.5. Sequence analysis of HsTNF-α 10
2.6. In vivo expression analysis of HsTNF-α 11
2.6.1. Tissue distribution of HsTNF-α mRNA 11
2.6.2. Expression profiles of HsTNF-α after NNV challenge 12
2.7. Construction of the HsTNF-α expression plasmid 14
2.8. Over-expression and purification of rHsTNF-α 15
2.9. SDS-PAGE 17
2.10. Western blotting assay 18
2.11. Cell viability 19
2.12. Preparation of plasmid DNA standards 20
2.13. Evaluation of rHsTNF-α activity 21
2.14. Statistical analysis 22
3. Results 23
3.1. The identification and bioinformatics analysis of HsTNF-α 23
3.2. Analysis of HsTNF-α mRNA expression in different tissues 29
3.3. Expression analysis of HsTNF-α in various tissues with NNV challenge 31
3.4. Production and purification of rHsTNF-α 33
3.5. Cytotoxicity assessment of rHsTNF-α 37
3.6. Antiviral effects of rHsTNF-α in SSN-1 cells 39
3.7. Evaluation of rHsTNF-α induced gene expression 43
4. Discussion 46
5. 국문 초록 53
6. References 55
- Degree
- Master
- Files in This Item:
-
-
There are no files associated with this item.
-
Items in Repository are protected by copyright, with all rights reserved, unless otherwise indicated.