题目: Characterization of LPS-induced TNFα factor (LITAF) from orange-spotted grouper, Epinephelus coioides 

作者: Jia Cai, Youhua Huang, Shina Wei, Zhengliang Ouyang, Xiaohong Huang*, Qiwei Qin*

刊物: Fish & Shellfish Immunology,（IF=2.964，I区）

刊号:  2013,35:1858-1866 

摘要: Lipopolysaccharide-induced TNFα factor (LITAF) is an important transcription factor that mediates cell apoptosis and inflammatory response. In the present study, we cloned and characterized a LITAF gene from orange-spotted grouper(Epinephelus coioides) (Ec-LITAF). Ec-LITAF encoded a predicted 142 amino acid protein which shared 74% identity to sablefish (Anoplopoma fimbria) LITAF homolog. Multiple amino acid alignment showed that Ec-LITAF contained a typicalLITAF domain with two CXXC motifs. Phylogenetic analysis indicated that Ec-LITAF was closely related to that of sablefish. Ec-LITAF mRNA was widely expressed in different tissues and its expression level in spleen was up-regulated after Singapore grouper iridovirus (SGIV) infection. Subcellular localization analysis revealed that the distribution of Ec-LITAFshowed diffuse and aggregated patterns in cytoplasm. Interestingly, the distribution of Ec-LITAF overlayed with a viral LITAFhomolog (vLITAF) encoded by SGIV. Overexpression of Ec-LITAF in vitro up-regulated the expression of tumor necrosis factors (TNF1 and TNF2) and TNF receptors (TNFR1 and TNFR2), and the expression of itself initiated apoptosis in fish cells. In addition, overexpression of Ec-LITAF not only accelerated SGIV infection induced CPE and cell death, but also increased viral gene transcription. Taken together, our data suggested that Ec-LITAF might play crucial roles during SGIV replication. 

题目: Molecular cloning, expression and functional analysis of ISG15 in orange-spotted grouper, Epinephelus coioides 

作者: Xiaohong Huang, Youhua Huang, Jia Cai, Shina Wei, Zhengliang Ouyang, Qiwei Qin* 

刊物: Fish & Shellfish Immunology,（IF=2.964，I区）

刊号:  2013,34(5):1094-1102 

摘要: Interferon-stimulated gene 15 (ISG15) is an ubiquitin homolog that is significantly induced by type I interferons or viral infections. Groupers, Epinephelus spp. being maricultured in China and Southeast Asian countries, always suffer from virus infection, including iridovirus and nodavirus. To date, the roles of grouper genes, especially interferon related genes in virus infection remained largely unknown. Here, the ISG15 homolog (EcISG15) was cloned from grouper Epinephelus coioides and its immune response to Singapore grouper iridovirus (SGIV) and grouper nervous necrosis virus (GNNV) was investigated. The full-length EcISG15 cDNA was composed of 948 bp and encoded a polypeptide of 155 amino acids with 37-68% identity with the known ISG15 homologs from other fish species. Amino acid alignment analysis indicated that EcISG15 contained two ubiquitin-like (UBL) domains and an Ub-conjugation domain (LRGG). Expressional analysis showed that EcISG15 was dramatically induced by GNNV infection, poly I:C or poly dA-dT treatment, but no obvious changes were observed during SGIV infection. Immunofluorescence assay showed that EcISG15 localized mainly in the cytoplasm of grouper cells in response to poly I:C stimulation or GNNV infection, but not in mock or SGIV infected cells. Western blot analysis indicated that the ISGylation was absent in SGIV-infected cells, but significantly enhanced in GNNV-infected or poly I:C transfected cells, suggesting that EcISG15 might play different roles in SGIV and GNNV infection. Furthermore, overexpression of EcISG15 in vitro inhibited the transcription of GNNV genes significantly. Taken together, the results indicated that fish ISG15might exert important roles against RNA virus infection. 

题目: Identification and characterization of TRP14, a thioredoxin-related protein of 14 kDa from orange-spotted grouper, Epinephelus coioides 

作者: Jingguang Wei, Huasong Ji, Minglan Guo, Yang Yan, Qiwei Qin* 

刊物: Fish & Shellfish Immunology,（IF=2.964，I区）

刊号:  2013,35:1670-1676 

摘要: 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-TRP14contained 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-TRP14could function as an important antioxidant in a physiological context, and might be involved in the responses to viral challenge. 

题目: Molecular cloning, characterization of one key molecule of teleost innate immunity from orange-spotted grouper (Epinephelus coioides):Serum amyloid A 

作者: Jingguang Wei, Minglan Guo, Huasong Ji, Qiwei Qin* 

刊物: Fish & Shellfish Immunology,（IF=2.964，I区）

刊号:  2013,34:296-304 

摘要: 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.