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1.
Fish Shellfish Immunol ; 83: 436-442, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30243776

RESUMO

Bcl-2 family proteins play essential roles in modulating immune response and controlling cells' fate. Bcl-xL is one of anti-apoptotic protein in this family. In this study, a new Bcl-xL homolog (EcBcl-xL) was identified and characterized from orange-spotted grouper, Epinephelus coioides. EcBcl-xL encoded a 221 amino acid peptides that shared 86% identity to Larimichthys crocea Bcl-xL protein, contained four conserved BH domains and one transmembrane region. The predicted three-dimensional structure of EcBcl-xL was similar with Homo sapiens Bcl-xL. EcBcl-xL widely expressed in all tested tissues with highest expression in head kidney. Its expression level was significantly up-regulated after SGIV infection in vivo. Furthermore, overexpression of EcBcl-xL could inhibit SGIV-induced nonapoptotic cell death and suppressed viral genes transcriptions in GS cells. Our findings suggested that EcBcl-xL might play a role during virus infection through modulating SGIV-induced nonapoptotic cell death.


Assuntos
Infecções por Vírus de DNA/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/imunologia , Iridovirus , Proteína bcl-X/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Morte Celular , Linhagem Celular , Infecções por Vírus de DNA/genética , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/genética , Proteínas de Peixes/genética , Peixes , Proteína bcl-X/genética
2.
Fish Shellfish Immunol ; 39(1): 61-8, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24811008

RESUMO

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the key adapter molecules in Toll-like receptor signal transduction that triggers downstream cascades involved in innate immunity. In the present study, a TRAF6 (named as Et-TRAF6) was identified from the marine fish grouper, Epinephelus tauvina by RACE PCR. The full-length cDNA of Et-TRAF6 comprised 1949 bp with a 1713 bp open reading frame (ORF) that encodes a putative protein of 570 amino acids. Similar to most TRAF6s, Et-TRAF6 includes one N-terminal RING domain (78aa-116aa), two zinc fingers of TRAF-type (159aa-210aa and 212aa-269aa), one coiled-coil region (370aa-394aa), and one conserved C-terminal meprin and TRAF homology (MATH) domain (401aa-526aa). Quantitative real-time PCR analysis revealed that Et-TRAF6 mRNA is expressed in all tested tissues, with the predominant expression in the stomach and intestine. The expression of Et-TRAF6 was up-regulated in the liver after challenge with Lipoteichoic acid (LTA), Peptidoglycan (PGN), Zymosan, polyinosine-polycytidylic acid [Poly(I:C)] and Polydeoxyadenylic acid · Polythymidylic acid sodium salt [Poly(dA:dT)]. The expression of Et-TRAF6 was also up-regulated in the liver after infection with Vibrio alginolyticus, Singapore grouper iridovirus (SGIV) and grouper nervous necrosis virus (GNNV). Recombinant Et-TRAF6 (rEt-TRAF6) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-Et-TRAF6 serum preparation. Intracellular localization revealed that Et-TRAF6 is distributed in both cytoplasm and nucleus, and predominantly in the cytoplasm. These results together indicated that Et-TRAF6 might be involved in immune responses toward bacterial and virus challenges.


Assuntos
Bass , Doenças dos Peixes/microbiologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Regulação da Expressão Gênica , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bass/classificação , Bass/genética , Bass/imunologia , Doenças dos Peixes/virologia , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/química , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo
3.
Fish Shellfish Immunol ; 35(5): 1670-6, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23994424

RESUMO

Thioredoxin (abbreviated as Trx) is an important ubiquitous disulfide reductase, which can protect organisms against various oxidative stresses. In the present study, a thioredoxin-related protein of 14 kDa (named as Ec-TRP14) was identified from the marine fish grouper, Epinephelus coioides by RACE PCR. The full-length cDNA of Ec-TRP14 was comprised of 1066 bp with a 372 bp open reading frame that encodes a putative protein of 123 amino acids. Similar to most TRP14s, Ec-TRP14 contained the conserved motif C-P-D-C. Ec-TRP14 mRNA is predominately expressed in liver, brain and muscle. The expression of Ec-TRP14 was up-regulated in the liver of grouper challenged with SGIV. Ec-TRP14 was recombined and expressed in Escherichia coli BL21 (DE3), and the rEc-Ec-TRP14 fusion protein was demonstrated to possess the antioxidant activity. The grouper spleen (GS) cells were treated with a high concentration of rEc-TRP14 (8.3 µg/ml), which significantly enhanced cells viability under damage caused by viral infection. These results together indicated that Ec-TRP14 could function as an important antioxidant in a physiological context, and might be involved in the responses to viral challenge.


Assuntos
Regulação da Expressão Gênica/imunologia , Perciformes/genética , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , Escherichia coli , Componentes do Gene , Regulação da Expressão Gênica/genética , Iridovirus/imunologia , Fígado/metabolismo , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Nefelometria e Turbidimetria , Perciformes/imunologia , Análise de Sequência de DNA
4.
Fish Shellfish Immunol ; 34(1): 296-304, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23178260

RESUMO

The orange-spotted grouper (Epinephelus coioides), a favorite marine food fish, is widely cultured in China and Southeast Asian countries. However, little is known about its acute phase response (APR) caused by viral diseases. Serum amyloid A (SAA) is a major acute phase protein (APP). In this study, a new SAA homologous (EcSAA) gene was cloned from grouper, E. coioides, by rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA sequence of SAA was 508 bp and contained a 363 bp open reading frame (ORF) coding for a protein of 121 aa. Similar to other fish known SAA genes, the EcSAA gene contained four exons and three introns. Quantitative real-time PCR analysis revealed that EcSAA mRNA is predominately expressed in liver and gill of grouper. Furthermore, the expression of EcSAA was differentially up-regulated in liver after infection with Staphyloccocus aureus, Vibrio vulnificus, Vibrio parahaemolyticus, Saccharomyces cerevisiae and Singapore grouper iridovirus (SGIV). Recombinant EcSAA (rEcSAA) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-EcSAA serum preparation. The rEcSAA fusion protein was demonstrated to bind to all tested bacteria and yeast, and inhibit the replication of SGIV. Overexpression of EcSAA in grouper spleen (GS) cells could also inhibit the replication of SGIV. These results suggest that EcSAA may be an important molecule in the innate immunity of grouper.


Assuntos
Bass/genética , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Proteína Amiloide A Sérica/genética , Sequência de Aminoácidos , Animais , Bactérias/imunologia , Infecções Bacterianas/enzimologia , Infecções Bacterianas/imunologia , Infecções Bacterianas/veterinária , Bass/imunologia , Clonagem Molecular , Infecções por Vírus de DNA/enzimologia , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , DNA Complementar/genética , Doenças dos Peixes/enzimologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/imunologia , Imunidade Inata , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , Reação em Cadeia da Polimerase/veterinária , RNA Mensageiro/genética , Ranavirus/imunologia , Saccharomyces cerevisiae/imunologia , Alinhamento de Sequência/veterinária , Análise de Sequência de DNA/veterinária , Proteína Amiloide A Sérica/imunologia
5.
Fish Shellfish Immunol ; 33(4): 916-25, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22986590

RESUMO

Translationally controlled tumor protein (TCTP) is an important molecule involved in multiple biological processes, such as cell growth, cell cycle progression, malignant transformation, and enhancement of the anti-apoptotic activity. In this study, the TCTP from orange-spotted grouper Epinephelus coioides (Ec-TCTP) was cloned and characterized. The full-length cDNA of Ec-TCTP was comprised of 1057 bp with a 510 bp open reading frame that encodes a putative protein of 170 amino acids. Recombinant Ec-TCTP (rEc-TCTP) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-Ec-TCTP serum preparation. The rEc-TCTP fusion protein was demonstrated to possess antioxidant activity, which conferred resistance to H(2)O(2) damage. Quantitative real-time PCR analysis revealed that Ec-TCTP mRNA is predominately expressed in the liver, and the expression was up-regulated in the liver of grouper after viral challenge with Singapore grouper iridovirus (SGIV). Intracellular localization revealed that Ec-TCTP expression was distributed predominantly in the cytoplasm. Although human TCTP has a role in apoptosis regulation, it is not known if grouper TCTP has any role in apoptosis regulation. Strikingly, grouper TCTP, when overexpressed in fathead minnow (FHM) cells, protected them from cell death induced by cycloheximide (CHX). In addition, overexpressed Ec-TCTP in grouper spleen (GS) cells inhibited the replication of SGIV. These results suggest that Ec-TCTP may play a critical role in their response to SGIV infection, through regulation of a cell death pathway that is common to fish and humans.


Assuntos
Bass/genética , Bass/imunologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Sequência de Aminoácidos , Animais , Antioxidantes/metabolismo , Antivirais/farmacologia , Apoptose , Bass/metabolismo , Bass/virologia , Biomarcadores Tumorais/química , Biomarcadores Tumorais/metabolismo , Linhagem Celular , Clonagem Molecular , Infecções por Vírus de DNA/imunologia , DNA Complementar/genética , Escherichia coli/genética , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica/veterinária , Regulação da Expressão Gênica , Injeções Intraperitoneais/veterinária , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , RNA Mensageiro/análise , Ranavirus , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência/veterinária , Análise de Sequência de DNA/veterinária , Proteína Tumoral 1 Controlada por Tradução
6.
Dev Comp Immunol ; 38(2): 254-61, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22885634

RESUMO

Vaccination is one of the best methods against viral diseases. In this study, experimental inactivated Singapore grouper iridovirus (SGIV) vaccines were prepared, and immunogenicity and protection against virus infection of the vaccines were investigated in orange-spotted grouper, Epinephelus coioides. Two kinds of vaccines, including ß-propiolactone (BPL) inactivated virus at 4°C for 12 h and formalin inactivated virus at 4°C for 12 d, was highly protective against the challenge at 30-day post-vaccination and produced relative percent of survival rates of 91.7% and 100%, respectively. These effective vaccinations induced potent innate immune responses mediated by pro-inflammatory cytokines and type I interferon (IFN)-stimulated genes (ISGs). It is noteworthy that ISGs, such as Mx and ISG15, were up-regulated only in the effective vaccine groups, which suggested that type I IFN system may be the functional basis of early anti-viral immunity. Moreover, effective vaccination also significantly up-regulated of the expression of MHC class I gene and produced substantial amount of specific serum antibody at 4 weeks post-vaccination. Taken together, our results clearly demonstrated that effective vaccination in grouper induced an early, nonspecific antiviral immunity, and later, a specific immune response involving both humoral and cell-mediated immunity.


Assuntos
Bass , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/imunologia , Iridovirus/fisiologia , Vacinas Virais/imunologia , Animais , Aquicultura , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/virologia , Doenças dos Peixes/virologia , Vacinas de Produtos Inativados/imunologia
7.
Fish Shellfish Immunol ; 33(3): 667-73, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22789714

RESUMO

Thioredoxin domain-containing protein 12 (Txndc12) belongs to the thioredoxin superfamily, and has roles in redox regulation, defense against oxidative stress, refolding of disulfide-containing proteins, and regulation of transcription factors. In this study, a thioredoxin domain-containing protein 12 was cloned from the marine fish grouper, Epinephelus coioides by RACE PCR, named as Ec-Txndc12. The Ec-Txndc12 encodes 173 amino acid residues with signal peptide in its N-terminal and a thioredoxin (Trx) domain that is homologous with some genes in Mus musculus, Xenopus laveis, etc. Ec-Txndc12 mRNA is predominately expressed in liver, brain and muscle. The expression of Ec-Txndc12 was up-regulated in the liver of grouper challenged with SGIV. In order to elucidate its biological functions, Ec-Txndc12 was recombined and expressed in Escherichia coli BL21 (DE3). The rEc-Txndc12 fusion protein was demonstrated to possess the antioxidant activity. The grouper spleen (GS) cells were treated with a high concentration of rEc-Txndc12 (30 µg/ml), which significantly enhanced cells viability under oxidative damage caused by viral infection. These results together indicated that Ec-Txndc12 could function as an important antioxidant in a physiological context, and might be involved in the responses to viral challenge.


Assuntos
Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Perciformes/genética , Perciformes/metabolismo , Proteína Dissulfeto Redutase (Glutationa)/genética , Proteína Dissulfeto Redutase (Glutationa)/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Proteínas de Peixes/química , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Dados de Sequência Molecular , Especificidade de Órgãos , Perciformes/imunologia , Perciformes/virologia , Filogenia , Proteína Dissulfeto Redutase (Glutationa)/química , Proteína Dissulfeto Redutase (Glutationa)/imunologia , RNA Mensageiro/análise , Ranavirus/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
8.
Dev Comp Immunol ; 38(1): 108-16, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22595860

RESUMO

Thioredoxins (TRXs) are a family of small, highly conserved proteins that are essential for the maintenance of cellular homeostasis. In this study, a thioredoxin gene was cloned from orange-spotted grouper, Epinephelus coioides (designated as Ec-TRX). The full-length cDNA of Ec-TRX was comprised of 767bp with a 327bp open reading frame that encodes a putative protein of 108 amino acids. Quantitative real-time PCR analysis revealed that the Ec-TRX mRNA was distributed abundantly in grouper, E. coioides skin and liver, and the expression in liver was up-regulated after viral challenge with Singapore grouper iridovirus (SGIV). Recombinant Ec-TRX (rEc-TRX) was expressed in Escherichia coli BL21 (DE3) and purified for mouse anti-Ec-TRX serum preparation. The rEc-TRX fusion protein was demonstrated to possess the expected redox activity in enzymatic analysis, and scavenge free radicals and protect supercoiled DNA from oxidative damage induced by a metal-ion catalyzed oxidation reaction. Subcellular localization revealed that Ec-TRX was distributed in both cytoplasm and nucleus. Overexpression of Ec-TRX in grouper spleen (GS) cells could promote the growth of GS cells and inhibit the replication of SGIV. These results suggest that Ec-TRX could function as an important antioxidant in a physiological context, and perhaps is involved in the responses to viral challenge.


Assuntos
Clonagem Molecular , Proteínas de Peixes/genética , Iridovirus , Tiorredoxinas/genética , Animais , Bass/genética , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/imunologia , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica , Filogenia , Tiorredoxinas/química , Tiorredoxinas/metabolismo
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