Expression pattern, promoter activity and bactericidal property of β-defensin from the mandarin fish Siniperca chuatsi.

β-Defensin (BD) are cysteine-rich, cationic antimicrobial peptides which play an important role in innate immune system against invading microbes. In the present study, the cDNA cloning, expression analysis, transcriptional regulation and antimicrobial activity of β-defensin (ScBD) from mandarin fish (Siniperca chuatsi) were characterized. The cDNA sequence of ScBD is 596 bp which encodes a protein of 63 amino acids (aa). The ScBD gene comprises three exons and two introns. The signal peptide is located in the first exon. ScBD contains 6 cysteines, and belongs to fish defensin 2 group based on phylogenetic analysis. Real-time quantitative PCR results showed that the mRNA transcripts of ScBD were distributed mainly in mucosal and lymphoid organs/tissues including intestine, gill, head kidney, kidney and spleen, with the highest level observed in spleen. Western blotting analysis revealed that the ScBD protein was abundant in head kidney, gill and spleen. A total of 3268 bp 5' flanking region of the ScBD gene promoter was sequenced, which contained a number of putative transcriptional binding sites for transcription factors. These transcription factors were analyzed using in vitro luciferase assay. The DNA region from position of -705 to -498 bp contains positive regulatory elements and that of -227 to +54 bp harbors the TATA which is essential for initiating gene expression. In addition, the ScBD peptide showed antibacterial activity against Escherichia coli M15, Staphylococcus aureus and Aeromonas hydrophila, whilst no effect on Edwardsiella tarda. These data suggest that the ScBD is importantly involved in host immune responses to invasion of bacterial pathogens.

Wang G ，Li J ，Zou P ，Xie H ，Huang B ，Nie P ，Chang M ... -  《-》

Β-defensin in Nile tilapia (Oreochromis niloticus): Sequence, tissue expression, and anti-bacterial activity of synthetic peptides.

Beta-defensins (β-defensins) are small cationic amphiphilic peptides that are widely distributed in plants, insects, and vertebrates, and are important for their antimicrobial properties. In this study, the β-defensin (Onβ-defensin) gene of the Nile tilapia (Oreochromis niloticus) was cloned from spleen tissue. Onβ-defensin has a genomic DNA sequence of 674 bp and produces a cDNA of 454 bp. Sequence alignments showed that Onβ-defensin contains three exons and two introns. Sequence analysis of the cDNA identified an open reading frame of 201 bp, encoding 66 amino acids. Bioinformatic analysis showed that Onβ-defensin encodes a cytoplasmic protein molecule containing a signal peptide. The deduced amino acid sequence of this peptide contains six conserved cysteine residues and two conserved glycine residues, and shows 81.82% and 78.33% sequence similarities with β-defensin-1 of fugu (Takifugu rubripes) and rainbow trout (Oncorhynchus mykiss), respectively. Real-time quantitative PCR showed that the level of Onβ-defensin expression was highest in the skin (307.1-fold), followed by the spleen (77.3-fold), kidney (17.8-fold), and muscle (16.5-fold) compared to controls. By contrast, low levels of expression were found in the liver, heart, intestine, stomach, and gill (<3.0-fold). Artificial infection of tilapia with Streptococcus agalactiae (group B streptococcus [GBS] strain) resulted in a significantly upregulated expression of Onβ-defensin in the skin, muscle, kidney, and gill. In vitro antimicrobial experiments showed that a synthetic Onβ-defensin polypeptide had a certain degree of inhibitory effect on the growth of Escherichia coli DH5α and S. agalactiae. The results indicate that Onβ-defensin plays a role in immune responses that suppress or kill pathogens.

Dong JJ ，Wu F ，Ye X ，Sun CF ，Tian YY ，Lu MX ，Zhang R ，Chen ZH ... -  《-》

Identification, expression and bioactivity of Paramisgurnus dabryanus β-defensin that might be involved in immune defense against bacterial infection.

β-defensins are a large family of multi-disulfide-bonded peptides with broad-spectrum antimicrobial activities that contribute to innate host defense in many organisms, but little information is available about β-defensins produced by freshwater fish lacking scales. We therefore cloned and identified a β-defensin gene from Chinese loach (Paramisgurnus dabryanus) by designing degenerate primers and using thermal asymmetric interlaced PCR. This gene is the first defensin gene ever identified in a non-scaled freshwater fish. Annotation of the protein domain architecture showed that the putative Chinese loach β-defensin contains the signature motif of six conserved cysteines within the mature peptide, an aspect similar to β-defensins of other marine fish. We also used quantitative real-time PCR to investigate the expression pattern of the Chinese loach β-defensin gene, mRNA of which could be observed in various tissues. After challenge with the pathogenic bacterium Aeromonas hydrophila, β-defensin expression was induced in the eye, gill, skin, and spleen of the adult loach. The bioactivity of the recombinant P. dabryanus β-defensin was examined against pathogenic bacteria, and the results suggest that this class 2 β-defensin has potential applications for treatment of bacterial infections.

Chen Y ，Zhao H ，Zhang X ，Luo H ，Xue X ，Li Z ，Yao B ... -  《-》

Metallothionein-2 gene from the mandarin fish Siniperca chuatsi: cDNA cloning, tissue expression, and immunohistochemical localization.

Gao D ，Wang GT ，Chen XT ，Nie P ... -  《COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C-TOXICOLOGY & PHARMACOLOGY》

Interferon regulatory factors 4 and 8 in rock bream, Oplegnathus fasciatus: structural and expressional evidence for their antimicrobial role in teleosts.

The interferon regulatory factor (IRF) members IRF4 and IRF8 contribute to B-lymphocyte development and can act as regulators of immunoglobulin (Ig) light chain gene transcription. These two IRFs are closely interrelated and are expressed at high levels in the lymphoid and myeloid cells of the immune system. In this study, the complete cDNA and genomic sequences of rock bream IRF4 (RbIRF4) and IRF8 (RbIRF8) were identified by homology screening of a multi-tissue normalized cDNA library and a BAC library, respectively, which had been established using Roche 454 GS-FLX™ technology. The full-length RbIRF4 cDNA is composed of 3442 bp and encodes a polypeptide of 462 amino acids; the genomic DNA is 9262 bp in length, consisting of eight exons and seven introns. The full-length RbIRF8 cDNA is composed of 2186 bp and encodes a 422 amino acid polypeptide; the genomic DNA is 4120 bp in length, consisting of nine exons and eight introns. The deduced amino acid sequences of RbIRF4 and RbIRF8 include a conserved DNA-binding domain (DBD) encompassing a tryptophan pentad-repeat and an IRF-association domain (IAD). Several putative transcription factor binding sites were also identified in 5' flanking region of both RbIRF4 and RbIRF8, and include those of immune-related factors. Quantitative real time PCR analysis of healthy rock bream detected the highest expression levels of RbIRF4 and RbIRF8 in lymphomyeloid-rich tissues. In addition, viral (rock bream iridovirus) and bacterial (Edwardsiella tarda and Streptococcus iniae) infection stimulated RbIRF4 and RbIRF8 expressions in head kidney and spleen. These results suggest not only that RbIRF4 and RbIRF8 may have a protective function against virus and bacteria pathogen invasion in rock bream, but also that IRFs may be immunomodulatory factors of teleost fish.

Bathige SD ，Whang I ，Umasuthan N ，Lim BS ，Park MA ，Kim E ，Park HC ，Lee J ... -  《-》