원문정보
초록
영어
CD63, a member of the tetraspanin family, is involved in the activation of immune cells, antiviral immunity, and signal transduction. The economically important anemonefishes Amphiprion sp. often face disease outbreaks, and the present study aimed to characterize CD63 in Amphiprion clarkii (denoted AcCD63) to enable better disease management. The in-silico analysis revealed that the AcCD63 transcript is 723 bp long and encodes 240 amino acids. The 26.2 kDa protein has a theoretical isoelectric point of 5.51. Similar to other tetraspanins, AcCD63 consists of four domains: short N-/C-terminal domains and small/large extracellular loops. Pairwise sequence alignment revealed that AcCD63 has the highest identity (100%) and similarity (99.2%) with CD63 from Amphiprion ocellaris. Multiple sequence alignment identified a conserved tetraspanin CCG motif, PXSCC motif, and C-terminal lysosome-targeting GYEVM motif. The quantitative polymerase chain reaction analysis showed that AcCD63 was highly expressed in the spleen and head kidney tissue, with low levels of expression in the liver. Temporal expression patterns of AcCD63 were measured in the head kidney and blood tissue after injection of polyinosinic:polycytidylic acid (poly (I:C)), lipolysacharides (LPS), or Vibrio harveyi (V. harveyi). AcCD63 was upregulated at 12 h post-injection with poly (I:C) or V. harveyi, and at 24 h post-injection with all stimulants in the head kidney. At 24 h post-injection, poly (I:C) and LPS upregulated, whereas V. harveyi downregulated AcCD63 expression in the blood. All viral hemorrhagic septicemia virus transcripts (M, G, N, RdRp, P, and NV) were downregulated in response to AcCD63 overexpression, and removal of viral particles occurred via the involvement of AcCD63. The expression of antiviral genes MX dynamin-like GTPase 1, interferon regulatory factor 3, interferon-stimulated gene 15, interferon-gamma, and viperin in CD63-overexpressing fathead minnow cells was downregulated. Collectively, our findings suggest that AcCD63 is an immunologically important gene involved in the A. clarkii pathogen stress response.
목차
1. Introduction
2. Materials and methods
2.1. AcCD63 sequence and bioinformatics analysis
2.2. Rearing of animals and challenge experiment
2.3. A. clarkii RNA extraction and cDNA synthesis
2.4. Spatial and temporal expression analysis
2.5. Plasmid construction for the viral assay
2.6. Cell culture conditions
2.7. Transfection of plasmids and viral infection
2.8. FHM cells and VHSV total RNA isolation and cDNA synthesis
2.9. qPCR of VHSV and FHM antiviral genes
2.10. Statistical analysis
3. Results
3.1. In-silico characterization of AcCD63
3.2. Spatial and temporal expression analysis
3.3. VHSV viral gene expression in AcCD63-overexpressing FHM cells
3.4. Mx, IRF3, ISG15, IFNγ, and Vip gene expression in AcCD63-overexpressing FHM cells
4. Discussion
References