Sequencing and expression analysis of CD3 gamma/delta and CD3E > chains in mandarin fish, Siniperca chuatsi

The genomic and cDNA sequences of the CD3 gamma/delta and CD3E > homologues in the mandarin fish, Siniperca chuats i, were determined. As in other vertebrate CD3 molecules, the deduced amino acid sequences of mandarin fish CD3 gamma/delta and CD3E > contained conserved residues and motifs, such as cysteine residues and CXXC and immunoreceptor tyrosine-based activation motifs. However, mandarin fish CD3 gamma/delta and CD3E > showed some differences to their mammalian counterparts, specifically the absence of a negatively charged residue in the transmembrane region of CD3 gamma/delta. Additionally, while an N -glycosylation site was present in CD3E >, the site was not observed in CD3 gamma/delta. The CD3 gamma/delta and CD3E > subunit sequences contain six and five exons, respectively, consistent with homologues from Atlantic salmon, Salmo salar. Phylogenetic analysis also revealed that CD3 gamma/delta and CD3E > in mandarin fish are closely related to their counterparts in Acanthopterygian fish. Real-time PCR showed CD3 gamma/delta and CD3E > were expressed mainly in the thymus and spleen in normal healthy fish and, to a lesser extent, in mucosal-associated lymphoid tissues, such as the intestine and gills. When lymphocytes isolated from head kidney were treated with the mitogens phytohemagglutinin, concanavalin, and polyriboinosinic polyribocytidylic acid, mRNA expression levels of CD3 gamma/delta and CD3E > were significantly elevated within 12 h of treatment. This indicated the presence of T lymphocytes in the head kidney of teleost fish, and also the recognition of mitogens by the lymphocytes. Mandarin fish infected with the bacterial pathogen Flavobacterium columnare also showed an increase in the expression of CD3 gamma/delta and CD3E > mRNA, indicating that CD3 gamma/delta and CD3E > lymphocytes are involved in the immune response of this species.The genomic and cDNA sequences of the CD3 gamma/delta and CD3E > homologues in the mandarin fish, Siniperca chuats i, were determined. As in other vertebrate CD3 molecules, the deduced amino acid sequences of mandarin fish CD3 gamma/delta and CD3E > contained conserved residues and motifs, such as cysteine residues and CXXC and immunoreceptor tyrosine-based activation motifs. However, mandarin fish CD3 gamma/delta and CD3E > showed some differences to their mammalian counterparts, specifically the absence of a negatively charged residue in the transmembrane region of CD3 gamma/delta. Additionally, while an N -glycosylation site was present in CD3E >, the site was not observed in CD3 gamma/delta. The CD3 gamma/delta and CD3E > subunit sequences contain six and five exons, respectively, consistent with homologues from Atlantic salmon, Salmo salar. Phylogenetic analysis also revealed that CD3 gamma/delta and CD3E > in mandarin fish are closely related to their counterparts in Acanthopterygian fish. Real-time PCR showed CD3 gamma/delta and CD3E > were expressed mainly in the thymus and spleen in normal healthy fish and, to a lesser extent, in mucosal-associated lymphoid tissues, such as the intestine and gills. When lymphocytes isolated from head kidney were treated with the mitogens phytohemagglutinin, concanavalin, and polyriboinosinic polyribocytidylic acid, mRNA expression levels of CD3 gamma/delta and CD3E > were significantly elevated within 12 h of treatment. This indicated the presence of T lymphocytes in the head kidney of teleost fish, and also the recognition of mitogens by the lymphocytes. Mandarin fish infected with the bacterial pathogen Flavobacterium columnare also showed an increase in the expression of CD3 gamma/delta and CD3E > mRNA, indicating that CD3 gamma/delta and CD3E > lymphocytes are involved in the immune response of this species

Characterization and expression of Cd8 molecules in mandarin fish Siniperca chuatsi

The full-length complementary DNA (cDNA) sequences encoding cd8a and cd8 beta molecules were sequenced and characterized from mandarin fish Siniperca chuatsi. Conserved motifs and residues were found to be present in derived peptides of the Cd8 molecules. For example, WXR motif, DXGXYXC motif, and four cysteine residues were present in the extracellular region of the Cd8 protein. Threonine, serine and proline residues involved in multiple O-linked glycosylation events were located in the membrane proximal hinge region. The common CPH motif in the cytoplasmic tail was detected similar to other teleost Cd8 molecules. Different from those in mammals, S. chuatsi Cd8 sequences have many extra cysteine residues (C149 in Cd8a sequence and C46, C51 and C158 in Cd8 beta sequence), which also exist in other teleost Cd8 molecules. Real-time polymerase chain reaction (RT-PCR) and Western blot analyses revealed that the thymus had the highest expression of cd8 messenger (m)RNA and protein. After stimulated with phytohaemagglutinin, polyriboinsine-polyribocyaidylic acid and concanavalin A (ConA), the expression level of cd8 mRNA increased significantly in head-kidney lymphocytes at 4 and 8 h, but decreased to normal level at 12 h. Similarly, stimulation with ConA in vivo also led to an increase in the cd8 mRNA level in the spleen. Immunohistochemistry analysis demonstrated that Cd8a-positive cells can be detected in the thymus, spleen and intestine by using polyclonal anti-Cd8a antibody.The full-length complementary DNA (cDNA) sequences encoding cd8a and cd8 beta molecules were sequenced and characterized from mandarin fish Siniperca chuatsi. Conserved motifs and residues were found to be present in derived peptides of the Cd8 molecules. For example, WXR motif, DXGXYXC motif, and four cysteine residues were present in the extracellular region of the Cd8 protein. Threonine, serine and proline residues involved in multiple O-linked glycosylation events were located in the membrane proximal hinge region. The common CPH motif in the cytoplasmic tail was detected similar to other teleost Cd8 molecules. Different from those in mammals, S. chuatsi Cd8 sequences have many extra cysteine residues (C149 in Cd8a sequence and C46, C51 and C158 in Cd8 beta sequence), which also exist in other teleost Cd8 molecules. Real-time polymerase chain reaction (RT-PCR) and Western blot analyses revealed that the thymus had the highest expression of cd8 messenger (m)RNA and protein. After stimulated with phytohaemagglutinin, polyriboinsine-polyribocyaidylic acid and concanavalin A (ConA), the expression level of cd8 mRNA increased significantly in head-kidney lymphocytes at 4 and 8 h, but decreased to normal level at 12 h. Similarly, stimulation with ConA in vivo also led to an increase in the cd8 mRNA level in the spleen. Immunohistochemistry analysis demonstrated that Cd8a-positive cells can be detected in the thymus, spleen and intestine by using polyclonal anti-Cd8a antibody

Construction of two selectable markers for integrative/conjugative plasmids in Flavobacterium columnare

Flavobacterium columnare, the etiological agent of columnaris disease, is one of the most important and widespread bacterial pathogens of freshwater fish. In this study, we constructed two artificial selectable markers (chloramphenicol and spectinomycin resistance) for gene transfer in F. columnare. These two new artificial selectable markers, which were created by placing the chloramphenicol or spectinomycin resistance gene under the control of the native acs regulatory region of F. columnare, were functional in both F. columnare and Escherichia coli. The integrative/conjugative plasmids constructed by using these markers were introduced into F. columnare G(4) via electroporation or conjugation. The integrated plasmid DNA was confirmed by Southern blotting and PCR analysis. These two markers can be employed in future investigations into gene deletion and the pathogenicity of virulence factors in F. columnare.Flavobacterium columnare, the etiological agent of columnaris disease, is one of the most important and widespread bacterial pathogens of freshwater fish. In this study, we constructed two artificial selectable markers (chloramphenicol and spectinomycin resistance) for gene transfer in F. columnare. These two new artificial selectable markers, which were created by placing the chloramphenicol or spectinomycin resistance gene under the control of the native acs regulatory region of F. columnare, were functional in both F. columnare and Escherichia coli. The integrative/conjugative plasmids constructed by using these markers were introduced into F. columnare G(4) via electroporation or conjugation. The integrated plasmid DNA was confirmed by Southern blotting and PCR analysis. These two markers can be employed in future investigations into gene deletion and the pathogenicity of virulence factors in F. columnare

Complementary DNA sequences of the constant regions of T-cell antigen receptors alpha, beta and gamma in mandarin fish, Siniperca chuatsi Basilewsky, and their transcriptional changes after stimulation with Flavobacterium columnare

In this study, the constant-region genes (C alpha, C beta and C gamma) that encode the T-cell antigen receptor (TCR) alpha, beta and gamma chains were cloned from mandarin fish, Siniperca chuatsi Basilewsky, an important freshwater fish species in China. The complementary DNA sequences of C alpha, C beta and C gamma were 843, 716 and 906base pairs (bp) in length and had a 465-, 289- and 360-bp 3 ' untranslated region, encoding 125, 142 and 182 amino acids, respectively. The amino-acid sequences of the constant regions of mandarin fish TCR alpha, beta and gamma chains (encoded by C alpha, C beta and C gamma, respectively) were most similar to those of their teleost counterparts, showing 60% similarity with pufferfish, 48% similarity with Atlantic salmon and 57% similarity with flounder, respectively. The phylogenetic analysis revealed that the mandarin fish C alpha, C beta and C gamma were clustered, respectively, with their vertebrate counterparts. The mandarin fish C alpha, C beta and C gamma could also be separated into four domains: immunoglobulin; connecting peptide (CP); transmembrane (TM); and cytoplasmic tail. Several conserved features in mammalian TCRs were also found in those of mandarin fish, such as a conserved cysteine residue in the CP domain of C alpha, necessary for creating an interchain disulphide bond with the TCR beta chain, and a conserved antigen receptor TM motif in C alpha and C beta. Meanwhile, transcripts of C alpha, C beta and C gamma were detectable in all examined organs, with a stronger signal observed in lymphoid organs. In addition, the temporal transcriptional changes for C alpha and C gamma were investigated, 1, 2, 3, 4, 5, 6 and 8weeks after stimulation with Flavobacterium columnare, in head kidney, spleen, blood, thymus, gill and intestine, using real-time polymerase chain reaction. The results demonstrated stimulation-dependent up-regulations in almost all tissues examined, which indicates that T cells may play important roles in preventing mandarin fish from bacterial invasion. In particular, apart from thymus, T cells were distributed mainly in gill and intestine, where striking up-regulation of C gamma was also observed. These results will facilitate functional studies of teleost TCRs and T cells.In this study, the constant-region genes (C alpha, C beta and C gamma) that encode the T-cell antigen receptor (TCR) alpha, beta and gamma chains were cloned from mandarin fish, Siniperca chuatsi Basilewsky, an important freshwater fish species in China. The complementary DNA sequences of C alpha, C beta and C gamma were 843, 716 and 906base pairs (bp) in length and had a 465-, 289- and 360-bp 3 ' untranslated region, encoding 125, 142 and 182 amino acids, respectively. The amino-acid sequences of the constant regions of mandarin fish TCR alpha, beta and gamma chains (encoded by C alpha, C beta and C gamma, respectively) were most similar to those of their teleost counterparts, showing 60% similarity with pufferfish, 48% similarity with Atlantic salmon and 57% similarity with flounder, respectively. The phylogenetic analysis revealed that the mandarin fish C alpha, C beta and C gamma were clustered, respectively, with their vertebrate counterparts. The mandarin fish C alpha, C beta and C gamma could also be separated into four domains: immunoglobulin; connecting peptide (CP); transmembrane (TM); and cytoplasmic tail. Several conserved features in mammalian TCRs were also found in those of mandarin fish, such as a conserved cysteine residue in the CP domain of C alpha, necessary for creating an interchain disulphide bond with the TCR beta chain, and a conserved antigen receptor TM motif in C alpha and C beta. Meanwhile, transcripts of C alpha, C beta and C gamma were detectable in all examined organs, with a stronger signal observed in lymphoid organs. In addition, the temporal transcriptional changes for C alpha and C gamma were investigated, 1, 2, 3, 4, 5, 6 and 8weeks after stimulation with Flavobacterium columnare, in head kidney, spleen, blood, thymus, gill and intestine, using real-time polymerase chain reaction. The results demonstrated stimulation-dependent up-regulations in almost all tissues examined, which indicates that T cells may play important roles in preventing mandarin fish from bacterial invasion. In particular, apart from thymus, T cells were distributed mainly in gill and intestine, where striking up-regulation of C gamma was also observed. These results will facilitate functional studies of teleost TCRs and T cells

Identification of immunogenic proteins of Flavobacterium columnare by two-dimensional electrophoresis immunoblotting with antibacterial sera from grass carp, Ctenopharyngodon idella (Valenciennes)

Flavobacterium columnare is a Gram-negative bacterium causing columnaris disease of freshwater fish worldwide, and development of efficacious vaccines has been a continuous challenge in aquaculture. In this study, 14 proteins were identified from cellular components of F.similar to columnare using an immunoblotting approach in two-dimensional electrophoresis map gels with antibacterial sera from grass carp, Ctenopharyngodon idella (Valenciennes), and then anti-grass carp-recombinant Ig (rIg) polyclonal antibodies. These proteins were characterized conclusively by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF/TOF MS). The 14 proteins are immunogenic molecules of F.similar to columnare, including chaperonins DnaK, GroEL and trigger factor, and translation elongation factor G, translation elongation factor Tu, 30S ribosomal subunit protein S1, dihydrolipoamide succinyltransferase, succinyl-CoA synthetase, SpoOJ regulator protein, alcohol dehydrogenase, fructose-bisphosphate aldolase, 3-hydroxybutyryl-CoA dehydrogenase and two conserved hypothetical proteins. These identified immunogenic proteins may provide candidate molecules for the development of vaccines against columnaris disease.Flavobacterium columnare is a Gram-negative bacterium causing columnaris disease of freshwater fish worldwide, and development of efficacious vaccines has been a continuous challenge in aquaculture. In this study, 14 proteins were identified from cellular components of F.similar to columnare using an immunoblotting approach in two-dimensional electrophoresis map gels with antibacterial sera from grass carp, Ctenopharyngodon idella (Valenciennes), and then anti-grass carp-recombinant Ig (rIg) polyclonal antibodies. These proteins were characterized conclusively by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF/TOF MS). The 14 proteins are immunogenic molecules of F.similar to columnare, including chaperonins DnaK, GroEL and trigger factor, and translation elongation factor G, translation elongation factor Tu, 30S ribosomal subunit protein S1, dihydrolipoamide succinyltransferase, succinyl-CoA synthetase, SpoOJ regulator protein, alcohol dehydrogenase, fructose-bisphosphate aldolase, 3-hydroxybutyryl-CoA dehydrogenase and two conserved hypothetical proteins. These identified immunogenic proteins may provide candidate molecules for the development of vaccines against columnaris disease

Roles of plasmid-encoded proteins, EseH, EseI and EscD in invasion, replication and virulence of Edwardsiella ictaluri

Native plasmids pEI1 and pEI2 were detected in Edwardsiella ictaluri HSN-1 isolated from diseased yellow catfish (Pelteobagrus fulvidraco). EseH encoded by pEI1 and other two proteins, EseI and EscD, encoded by pEI2, were found with homology to type III secretion system (T3SS) proteins. To investigate their roles in pathogenesis, the native plasmids were cured based on plasmid incompatibility by introducing a Kan positive and SacB negative selection marker into gene spacer of the native plasmids. Mutants with the deletion of the target genes were obtained by reverse PCR and self-ligation, and all mutants were examined for their virulence effect in yellow catfish. Compared with the HSN-1 strain, the two mutants Delta eseH and Delta eseI were attenuated, while mutant Delta escD had increased virulence with higher Competitive Index (CI) value. The adherence and invasion assays on fish EPC cells indicated that Delta eseH and Delta eseI had decreased ability in adherence. Using E. tarda as surrogate, EseH and EseI were detected in culture supernatants, but EscD was not, with the secretion of EseH depending on T3SS. In addition, EseH and EseI were found translocated into host cells, and by means of subcellular fractionation, EseH was localized in membrane fraction of ZF4 cells, and EseI in the cytosol fraction. Hence, the role of these three genes in adherence, invasion and cellular replication was revealed from the pathogenic bacterium E. ictaluri. (C) 2013 Elsevier B.V. All rights reserved.Native plasmids pEI1 and pEI2 were detected in Edwardsiella ictaluri HSN-1 isolated from diseased yellow catfish (Pelteobagrus fulvidraco). EseH encoded by pEI1 and other two proteins, EseI and EscD, encoded by pEI2, were found with homology to type III secretion system (T3SS) proteins. To investigate their roles in pathogenesis, the native plasmids were cured based on plasmid incompatibility by introducing a Kan positive and SacB negative selection marker into gene spacer of the native plasmids. Mutants with the deletion of the target genes were obtained by reverse PCR and self-ligation, and all mutants were examined for their virulence effect in yellow catfish. Compared with the HSN-1 strain, the two mutants Delta eseH and Delta eseI were attenuated, while mutant Delta escD had increased virulence with higher Competitive Index (CI) value. The adherence and invasion assays on fish EPC cells indicated that Delta eseH and Delta eseI had decreased ability in adherence. Using E. tarda as surrogate, EseH and EseI were detected in culture supernatants, but EscD was not, with the secretion of EseH depending on T3SS. In addition, EseH and EseI were found translocated into host cells, and by means of subcellular fractionation, EseH was localized in membrane fraction of ZF4 cells, and EseI in the cytosol fraction. Hence, the role of these three genes in adherence, invasion and cellular replication was revealed from the pathogenic bacterium E. ictaluri. (C) 2013 Elsevier B.V. All rights reserved

Establishment of a Cell Line from Chinese Soft-shelled Turtle Pelodiscus sinensis with the Practicability of Transfection and Viral Replication

continuous cell line derived from extracranial carotid artery of the Chinese soft-shelled turtle Pelodiscus sinensis was established and characterized. It was named as STA (soft-shelled turtle artery) cell line. The cells were muscle-cell like, and were stained with antibody against smooth muscle myofilament marker, a-smooth muscle actin. These cells had a normal diploid chromosome number of 2n = 66, and their 12S rRNA and 16S rRNA sequences shared 99% identity with ones of the turtle in database. The cell line can be cultured well in media DMEM/F12 or M199 supplemented with 10% FBS at 28 C. It was further demonstrated that the cells were transfected successfully with pTurbo plasmid, with a transfection efficiency being over 30%. The soft-shelled turtle iridovirus (STIV) propagated in the cell line, causing typical CPE with the formation of inclusion bodies. It is suggested that the established STA cell line provides a convenient platform for studying the pathogenesis of STIV and biological aspects of the turtle.continuous cell line derived from extracranial carotid artery of the Chinese soft-shelled turtle Pelodiscus sinensis was established and characterized. It was named as STA (soft-shelled turtle artery) cell line. The cells were muscle-cell like, and were stained with antibody against smooth muscle myofilament marker, a-smooth muscle actin. These cells had a normal diploid chromosome number of 2n = 66, and their 12S rRNA and 16S rRNA sequences shared 99% identity with ones of the turtle in database. The cell line can be cultured well in media DMEM/F12 or M199 supplemented with 10% FBS at 28 C. It was further demonstrated that the cells were transfected successfully with pTurbo plasmid, with a transfection efficiency being over 30%. The soft-shelled turtle iridovirus (STIV) propagated in the cell line, causing typical CPE with the formation of inclusion bodies. It is suggested that the established STA cell line provides a convenient platform for studying the pathogenesis of STIV and biological aspects of the turtle

IFN-gamma and its receptors in a reptile reveal the evolutionary conservation of type II IFNs in vertebrates

In this study, interferon gamma (IFN-gamma) and interferon gamma receptor (IFN-gamma R) genes have been identified in non-avian reptile, the North American green anole lizard (Anolis carolinensis). Like their counterparts from other jawed vertebrates, lizard IFN-gamma, IFN-gamma R1 and IFN-gamma R2 show conserved features in genomic organizations, gene loci and protein sequences. The IFN-gamma gene has the full cDNA sequence of 936 bp, with 522 bp open reading frame (ORF) encoding 174 amino acids, and has the genomic organization of four exons and three introns as observed in IFN-gamma genes of other classes of vertebrates. The receptors, IFN-gamma R1 and IFN-gamma R2 have the ORF of 1278 and 984 bp, coding for 425 and 327 aa, respectively, with the genome organization of seven exons and six introns. In the gene loci of IFN-gamma, DYRK2, IL22, IL26 and MDM1 are found with conserved synteny in vertebrates, and similar genes adjacent to IFN-gamma R1 and IFN-gamma R2 were also found. These receptors also contain conserved motifs, such as the membrane-proximal region and the C-terminal five residue motif in IFN-gamma R1, and intracellular conservative sequence in IFN-gamma R2, which have been confirmed to mediate down-stream JAK-STAT signaling pathway in mammals. IFN-gamma and its receptors, IFN-gamma R1 and IFN-gamma R2 were constitutively expressed in organs/tissues examined in the lizard, and up-regulated expression of IFN-gamma was observed in organs/tissues examined following the poly(I:C) stimulation, suggesting its antiviral role in lizards. The conserved features of IFN-gamma and its receptors, IFN-gamma R1 and IFN-gamma R2, in gene organization and gene locus as well as in functional domain or motif may imply that the function of type II IFN system is evolutionarily conserved in the green anole lizard, as observed in other classes of vertebrates. (C) 2013 Elsevier Ltd. All rights reserved.In this study, interferon gamma (IFN-gamma) and interferon gamma receptor (IFN-gamma R) genes have been identified in non-avian reptile, the North American green anole lizard (Anolis carolinensis). Like their counterparts from other jawed vertebrates, lizard IFN-gamma, IFN-gamma R1 and IFN-gamma R2 show conserved features in genomic organizations, gene loci and protein sequences. The IFN-gamma gene has the full cDNA sequence of 936 bp, with 522 bp open reading frame (ORF) encoding 174 amino acids, and has the genomic organization of four exons and three introns as observed in IFN-gamma genes of other classes of vertebrates. The receptors, IFN-gamma R1 and IFN-gamma R2 have the ORF of 1278 and 984 bp, coding for 425 and 327 aa, respectively, with the genome organization of seven exons and six introns. In the gene loci of IFN-gamma, DYRK2, IL22, IL26 and MDM1 are found with conserved synteny in vertebrates, and similar genes adjacent to IFN-gamma R1 and IFN-gamma R2 were also found. These receptors also contain conserved motifs, such as the membrane-proximal region and the C-terminal five residue motif in IFN-gamma R1, and intracellular conservative sequence in IFN-gamma R2, which have been confirmed to mediate down-stream JAK-STAT signaling pathway in mammals. IFN-gamma and its receptors, IFN-gamma R1 and IFN-gamma R2 were constitutively expressed in organs/tissues examined in the lizard, and up-regulated expression of IFN-gamma was observed in organs/tissues examined following the poly(I:C) stimulation, suggesting its antiviral role in lizards. The conserved features of IFN-gamma and its receptors, IFN-gamma R1 and IFN-gamma R2, in gene organization and gene locus as well as in functional domain or motif may imply that the function of type II IFN system is evolutionarily conserved in the green anole lizard, as observed in other classes of vertebrates. (C) 2013 Elsevier Ltd. All rights reserved

IFN-gamma in turtle: Conservation in sequence and signalling and role in inhibiting iridovirus replication in Chinese soft-shelled turtle Pelodiscus sinensis

The IFN-gamma gene was identified in a turtle, the Chinese soft-shelled turtle, Pelodiscus sinensis, with its genome consisting of 4 exons and 3 introns. The deduced amino acid sequence of this gene contains a signal peptide, an IFN-gamma family signature motif (130)IQRKAVNELFPT, an NLS motif (KRKR)-K-155 and three potential N-glycosylation sites. As revealed by real-time quantitative PCR, the gene was constitutively expressed in all tested organs/tissues, with higher level observed in blood, intestine and thymus. An induced expression of IFN-gamma at mRNA level was observed in peripheral blood leucocytes (PBLs) in response to in vitro stimulation of LPS and PolyI:C. The overexpression of IFN-gamma in the Chinese soft-shelled turtle artery (STA) cell line resulted in the increase in the expression of transcriptional regulators, such as IRF1, IRF7 and STAT1, and antiviral genes, such as Mx, PKR, implying possibly the existence of a conserved signalling network and role for IFN-gamma in the turtle. Furthermore, the infection of soft-shelled turtle iridovirus (STIV) in the cell line transfected with IFN-gamma may cause the cell death as demonstrated with the elevated lactate dehydrogenase (LDH) level and cell mortality. However, the mechanism involved in the antiviral activity may require further investigation. (C) 2013 Elsevier Ltd. All rights reserved.The IFN-gamma gene was identified in a turtle, the Chinese soft-shelled turtle, Pelodiscus sinensis, with its genome consisting of 4 exons and 3 introns. The deduced amino acid sequence of this gene contains a signal peptide, an IFN-gamma family signature motif (130)IQRKAVNELFPT, an NLS motif (KRKR)-K-155 and three potential N-glycosylation sites. As revealed by real-time quantitative PCR, the gene was constitutively expressed in all tested organs/tissues, with higher level observed in blood, intestine and thymus. An induced expression of IFN-gamma at mRNA level was observed in peripheral blood leucocytes (PBLs) in response to in vitro stimulation of LPS and PolyI:C. The overexpression of IFN-gamma in the Chinese soft-shelled turtle artery (STA) cell line resulted in the increase in the expression of transcriptional regulators, such as IRF1, IRF7 and STAT1, and antiviral genes, such as Mx, PKR, implying possibly the existence of a conserved signalling network and role for IFN-gamma in the turtle. Furthermore, the infection of soft-shelled turtle iridovirus (STIV) in the cell line transfected with IFN-gamma may cause the cell death as demonstrated with the elevated lactate dehydrogenase (LDH) level and cell mortality. However, the mechanism involved in the antiviral activity may require further investigation. (C) 2013 Elsevier Ltd. All rights reserved

Three goose-type lysozymes in the gastropod Oncomelania hupensis: cDNA sequences and lytic activity of recombinant proteins

Three goose-type (g-type) lysozymes, designated as OHLysG1, OHLysG2 and OHLysG3 were identified from expressed sequence tags (ESTs) of a gastropod Oncomelania hupensis, the intermediate host of Schistosoma japonicum. The full cDNA sequences of OHLysG1, OHLysG2 and OHLysG3 consisted of 735, 909 and 808 nucleotides, with an open reading frame of 198, 214 and 249 codons containing a 21, 7 and 8 amino acid (aa) signal peptide at the N-terminus, respectively. The three g-type lysozymes shared conserved features with other g-type lysozymes, such as the substrate binding sites, the catalytic residues critical for the fundamental structure and function of g-type lysozymes. It seems possible that g-type lysozymes in molluscs shared one conserved cysteine with those in birds and mammals, and six conserved cysteines were observed for mollusc g-type lysozymes, with two unique cysteines present in the g-type lysozymes of O. hupensis. The three lysozyme genes were expressed mainly in hepatopancreas, with relatively low expression level observed in head-foot muscle and intestine. When comparing S. japonicum-infected and uninfected snails, significant increase (P &lt; 0.05) was observed for all the three lysozymes in infected snails, with the highest increase detected in hepatopancreas, and lowest in intestine, implying their defensive role in the host-parasite, i.e. snail-trematode system. The three recombinant lysozymes expressed in Escherichia coil strain M15 showed lytic activity against Aeromonas hydrophila, Vibrio fluvialis, Aeromonas sobria and Micrococcus lysodeikticus. In conclusion, the finding of three g-type lysozymes in O. hupensis provides structural and functional evidence of multiple g-type lysozymes in gastropod, which may have evolutional implication in the snail-trematode system. (C) 2011 Elsevier Ltd. All rights reserved.Three goose-type (g-type) lysozymes, designated as OHLysG1, OHLysG2 and OHLysG3 were identified from expressed sequence tags (ESTs) of a gastropod Oncomelania hupensis, the intermediate host of Schistosoma japonicum. The full cDNA sequences of OHLysG1, OHLysG2 and OHLysG3 consisted of 735, 909 and 808 nucleotides, with an open reading frame of 198, 214 and 249 codons containing a 21, 7 and 8 amino acid (aa) signal peptide at the N-terminus, respectively. The three g-type lysozymes shared conserved features with other g-type lysozymes, such as the substrate binding sites, the catalytic residues critical for the fundamental structure and function of g-type lysozymes. It seems possible that g-type lysozymes in molluscs shared one conserved cysteine with those in birds and mammals, and six conserved cysteines were observed for mollusc g-type lysozymes, with two unique cysteines present in the g-type lysozymes of O. hupensis. The three lysozyme genes were expressed mainly in hepatopancreas, with relatively low expression level observed in head-foot muscle and intestine. When comparing S. japonicum-infected and uninfected snails, significant increase (P < 0.05) was observed for all the three lysozymes in infected snails, with the highest increase detected in hepatopancreas, and lowest in intestine, implying their defensive role in the host-parasite, i.e. snail-trematode system. The three recombinant lysozymes expressed in Escherichia coli strain M15 showed lytic activity against Aeromonas hydrophila, Vibrio fluvialis, Aeromonas sobria and Micrococcus lysodeikticus. In conclusion, the finding of three g-type lysozymes in O. hupensis provides structural and functional evidence of multiple g-type lysozymes in gastropod, which may have evolutional implication in the snail-trematode system. (C) 2011 Elsevier Ltd. All rights reserved