Identification and characterization of a Cystatin gene from Chinese mitten crab Eriocheir sinensis

Cystatins are a superfamily of proteins as reversible inhibitor of cysteine proteinases which play essential roles in a spectrum of physiological and immunological processes In this study, a novel member of Cystatin superfamily was identified from Chinese mitten crab Enocheir sinensis (designated EsCystain) by expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approaches The full-length cDNA of EsCystatin was of 1486 bp, consisting of a 5'-terminal untranslated region (UTR) of 92 bp, a 3' UTR of 1034 bp with a polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 360 bp encoded a polypeptide of 120 amino acids with the theoretical isoelectric point of 548 and the predicted molecular weight of 13 39 kDa. A signal Cystatin-like domain (Gly(25) to Lys(112)) was found in the putative amino acid sequences of EsCystatin Similar to other Cystatins, the conserved central Q(70)VVSG(74) motif was located in the Cystatin-like domain of EsCystatin But EsCystatin lacked of signal peptide and disulphide bond. The EsCystatin exhibited homology with the other known Cystatins from invertebrates and higher vertebrates, and it was clustered into Cystatin family 1 in the phylogenetic tree. The mRNA transcripts of EsCystain were mainly expressed in hemolymph, gill, hepatopancreas, gonad and muscle, and also marginally detectable in heart After Listonella anguillarum challenge, the relative expression level of EsCystatin in hemolymph was down-regulated to 0 6-fold (P < 0.05) at 3 h post-challenge. Subsequently, it was up-regulated to 3.0-fold (P < 0.01)at 24 h Afterwards. EsCystatin mRNA transcripts suddenly decreased to original level. After Pichia pastoris GS115 challenge, its mRNA expression level in hemolymph was up-regulated to the peak at 3 h (2 8-fold of that in blank (P < 0 01)) The cDNA fragment encoding the mature peptide of EsCystatin was recombined and expressed in Escherichia coli Rosetta-gami (DE3). The recombinant EsCystatin displayed a promoter inhibitory activity against papain When the concentration of EsCystatin protein was of 300 mu g mL(-1), almost 89% of papain activity could be inhibited. These results collectively suggested that EsCystatin was a novel member of protein in Cystatin family, was a potent inhibitor of papain and involved in immune response versus invading microorganisms. (C) 2010 Elsevier Ltd All rights reserved.

Identification and molecular analysis of a stress-inducible Hsp70 from Sciaenops ocellatus

Hsp70 proteins are a family of molecular chaperones that are involved in many aspects of protein homeostasis. In this study, an Hsp70 homologue (SoHsp70) was identified from red drum Sciaenops ocellatus and analyzed at molecular level. The open reading frame of SoHsp70 is 1920 bp and intronless, with a 5'-untranslated region (UTR) of 399 bp and a 3'-UTR of 241 bp. The deduced amino acid sequence of SoHsp70 shares 84-92% overall identities with the Hsp70s of a number of fish species. In silico analysis identified in SoHsp70 three conserved Hsp70 domains involved in nucleotide and substrate binding. The coding sequence of SoHsp70 was subcloned into Escherichia coli, from which recombinant SoHsp70 was purified and, upon ATPase assay, found to exhibit apparent ATPase activity. Expressional analysis showed that constitutive expression of SoHsp70 was detectable in heart, liver, spleen, kidney, brain, blood, and gill. Experimental challenges with poly(I:C) and bacterial pathogens of Gram-positive and Gram-negative nature induced SoHsp70 expression in kidney to different levels. Stress-responsive analysis of SoHsp70 expression in primary cultures of red drum hepatocytes showed that acute heat shock treatment elicited a rapid induction of SoHsp70 expression which appeared after 10 min and 30 min of treatment. Exposure of hepatocytes separately to iron, copper, mercury, and hydrogen peroxide significantly unregulated SoHsp70 expression in time-dependent manners. Vaccination of red drum with a Streptococcus iniae bacterin was also found to induce SoHsp70 expression. Furthermore, recombinant SoHsp70 enhanced the immunoprotective effect of a subunit vaccine. Taken together, these results suggest that SoHsp70 is a stress-inducible protein that is likely to play a role in immunity and in coping with environmental and biological stresses. (C) 2010 Elsevier Ltd. All rights reserved.

Identification of an Edwardsiella tarda surface antigen and analysis of its immunoprotective potential as a purified recombinant subunit vaccine and a surface-anchored subunit vaccine expressed by a fish commensal strain

Edwardsiella tarda is the etiological agent of edwardsiellosis, a systematic disease that affects a wide range of marine and freshwater fish cultured worldwide. In order to identify E. tarda antigens with vaccine potential, we in this study conducted a systematic search for E. tarda proteins with secretion capacity. One of the proteins thus identified was Esa1, which contains 795 amino acid residues and shares extensive overall sequence identities with the D15-like surface antigens of several bacterial species. In silico analyses indicated that Esa1 localizes to outer membrane and possesses domain structures that are conserved among bacterial surface antigens. The vaccine potential of purified recombinant Esa1 was examined in a Japanese flounder (Paralichthys olivaceus) model, which showed that fish vaccinated with Esa1 exhibited a high level of survival and produced specific serum antibodies. Passive immunization of naive fish with antisera raised against Esa1 resulted in significant protection against E. tarda challenge. Taking advantage of the secretion capacity of Esa1 and the natural gut-colonization ability of a fish commensal strain, we constructed an Esa1-expressing recombinant strain, FP3/pJsa1. Western immunoblot and agglutination analyses showed that FP3/pJsa1 produces outer membrane-localized Esa1 and forms aggregates in the presence of anti-Esa1 antibodies. Vaccination analyses showed that FP3/pJsa1 as an intraperitoneal injection vaccine and an oral vaccine embedded in alginate microspheres produced relative percent survival rates of 79% and 52%, respectively, under severe challenging conditions that resulted in 92-96% mortality in control fish. Further analyses showed that following oral vaccination, FP3/pJsa1 was able to colonize in the gut but unable to disseminate into other tissues. Together these results indicate that Esa1 is a protective immunogen and an effective oral vaccine when delivered by FP3/pJsa1 as a surface-anchored antigen. (c) 2010 Elsevier Ltd. All rights reserved.

Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity

Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in certain contexts are known to be immunostimulatory in vertebrate systems. CpG ODNs with immune effects have been identified for many fish species but, to our knowledge, not for turbot. In this study, a turbot-effective CpG ODN, ODN 205, was identified and a plasmid, pCN5, was constructed which contains the CpG motif of ODN 205. When administered into turbot via intraperitoneal (i.p.) injection, both ODN 205 and pCN5 could (i) inhibit bacterial dissemination in blood in dose and time dependent manners, and (ii) protect against lethal bacterial challenge. Immunological analyses showed that in vitro treatment with ODN 205 stimulated peripheral blood leukocyte proliferation, while i.p. injection with ODN 205 enhanced the respiratory burst activity, chemiluminescence response, and acid phosphatase activity of turbot head kidney macrophages. pCN5 treatment-induced immune responses similar to those induced by ODN 205 treatment except that pCN5 could also enhance serum bactericidal activity in a calcium-independent manner. To examine whether ODN 205 and pCN5 had any effect on specific immunity, ODN 205 and pCN5 were co-administered into turbot with a Vibrio harveyi subunit vaccine, DegQ. The results showed that pCN5, but not ODN 205, significantly increased the immunoprotective efficacy of DegQ and enhanced the production of specific serum antibodies in the vaccinated fish. Further analysis indicated that vaccination with DegQ in the presence of pCN5 upregulated the expression of the genes encoding MHC class II alpha, IgM, Mx, and IL-8 receptor. Taken together, these results demonstrate that ODN 205 and pCN5 can stimulate the immune system of turbot and induce protection against bacterial challenge. In addition, pCN5 also possesses adjuvant property and can potentiate vaccine-induced specific immunity. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and analysis of a Sciaenops ocellatus ISG15 homologue that is involved in host immune defense against bacterial infection

ISG15 is an interferon-stimulated gene that encodes a ubiquitin-like protein. ISG15 homologues have been identified in a number of fish species, some of which are known to be regulated at expression level by virus infection and lipopolysacchande (LPS) treatment However, the relationship between ISG15 and live bacterial infection has not been investigated in piscine models. In this study, an ISG15 homologue, SoISG15, was identified from red drum Scraeriops ocellaws and analyzed at expression and functional levels The open reading frame ofSolSG15 is 477 base pairs (bp) and mtronless, with a 5'-untranslated region (UTR) of 91 bp and a 3'-UTR of 415 bp The deduced amino acid sequence of S0ISG15 shares 60-67% overall identities with the ISG15 of several fish species. S0ISG15 possesses two conserved ubiquinn-like domains and the canonical ubiquitin conjugation motif, LRGG, at the C-terminus. Expressional analysis showed that constitutive expression of SolSG15 was highest in blood and lowest in kidney Experimental challenges with LPS and bacterial pathogens induced significant S0ISG15 expression in the kidney but not in the liver Similar differential induction was also observed at cellular level with primary hepatocytes and head kidney (HK) lymphocytes. Poly(' C), however, effected drastic induction of S0ISG15 expression in kidney and liver at both tissue and cellular levels. Immunoblot analysis showed that S0ISG15 was secreted by cultured HK lymphocytes into the extracellular milieu. Recombinant S0ISG15 expressed in and purified from Eschenclua colt was able to enhance the respiratory burst activity, acid phosphatase activity, and bactericidal activity of HK macrophages. Taken together, the results of this study indicated that SoISG 15 possesses apparent immunological property and is likely to be involved in host immune defense against bacterial infection. (C)2010 Elsevier Ltd All rights reserved.

The genetic analysis and germplasm identification of the gametophytes of Undaria pinnatifida (Phaeophyceae) with RAPD method

Eleven pairs of Undaria pinnatifida (Harv.) Suringar gametophytes were identified with random amplified polymorphic DNA (RAPD) technique. After screening 100 primers, 20 ten-base primers were determined for the RAPD analysis. A total of 312 polymorphic loci were obtained, of which 97.7% were polymorphic. The primer S198 was found to distinguish all the selected Undaria pinnatifida gametophytes. The genetic distances between each two of the twenty-two U. pinnatifida gametophytes ranged from 0.080 to 0.428, while the distances to the Laminaria was 0.497 on average. After reexamination, two sequences characterized amplification region (SCAR) markers were successfully converted, which could be applied to U. pinnatifida germplasm identification. All these results demonstrated the feasibility of applying RAPD markers to germplasm characterization and identification of U. pinnatifida gametophytes, and to provide a molecular basis for Undaria breeding.

Cloning, expression and identification of ferritin from Chinese shrimp, Fenneropenaeus chinensis

Ferritin, the iron storage protein, plays a key role in iron metabolism. A cDNA encoding ferritin (FcFer) was cloned from hepatopancreas of Chinese shrimp, Fenneropenaeus chinensis. The predicted protein contains 170 amino acid residues with a predicted molecular weight (MW) about 19, 422.89 Da and theoretical isoelectric point (PI) of 4.73. Amino acid alignment of FcFer revealed 97% homology with Litopenaeus vannamei ferritin. Results of the RT-PCR showed that the expression of FcFer mRNA was up-regulated after shrimp was challenged with either white spot syndrome virus (WSSV) or heavy metal ions (Zn2+ and Cu2+) in the laboratory. A fusion protein containing FcFer was produced and the purified recombinant protein exhibited similar function of iron uptake in vitro. The result of in-gel digestion and identification using LC-ESI-MS showed that two peptide fragments (-DDVALPGFAK- and -LLEDEYLEEQVDS1KK-) of the recombinant protein were identical to the corresponding sequence of L. vannamei ferritin. The recombinant FcFer protein will be proved useful for study on the structure and function of ferritin in F chinensis. (c) 2006 Elsevier B.V. All rights reserved.

Identification and molecular analysis of a novel C-type lectin from Scophthalmus maximus

C-type lectins are calcium-dependent carbohydrate-binding proteins that play Important roles in innate immunity In this study, a C-type lectin homologue (SmLec1) was identified from turbot (Scophthalmus maximus) and analyzed at expression and functional levels. The open reading frame of SmLec1 is 504 bp, with a 5'-untranslated region (UTR) of 101 bp and a 3'-UTR of 164 bp The deduced amino acid sequence of SmLec1 shares 34%-38% overall identities with the C-type lectins of several fish species In silico analysis identified in SmLec1 conserved C-type lectin features, including a carbohydrate-recognition domain, four disulfide bond-forming cysteine residues, and the mannose-type carbohydrate-binding motif In addition, SmLec1 possesses a putative signal peptide sequence and is predicted to be localized in the extracellular. Expression of SmLec1 was highest in liver and responded positively to experimental challenges with fish pathogens Recombinant SmLec1 (rSmLec1) purified from yeast was able to agglutinate the Gram-negative fish pathogen Listonella anguillarum but not the Gram-positive pathogen Streptococcus uncle The agglutinating ability of rSmLec1 was abolished in the presence of mannose and ethylenediaminetetraacetic acid and by elevated temperature (65 degrees C) Further analysis showed that rSmLec1 could stimulate kidney lymphocyte proliferation and enhance the killing of bacterial pathogen by macrophages Taken together, these results suggest that SmLec1 is a unique mannose-binding C-type lectin that possesses apparent immunomodulating property and is likely to be involved in host defense against bacterial infection (C) 2010 Elsevier Ltd. All rights reserved

Species identification in salted products of red snappers by semi-nested PCR-RFLP based on the mitochondrial 12S rRNA gene sequence

A molecular approach was developed to distinguish species of red snappers among commercial salted fish products. The specific fragments of the mitochondrial 12S rRNA gene, which were about 450bp, were obtained using the semi-nested polymerase chain reaction (semi-nested PCR). Subsequently, PCR arnplicons were sequenced, aiming to select restriction endonucleases that generated species-specific restriction fragment length polymorphism (RFLP) profiles. Discrimination of red snappers Lutjanus sanguineus, Lutjanus erythopterus from Lutjanus argentimaculatus, Lutjanus malabarius and other morphologically similar fishes such as Lethrinus leutjanus and Pinjalo pinjalo was feasible by one restriction digestion reaction with three endonucleases Hae III, Sca I and SnaB I, however, for discrimination of L. sanguineus and L. erythopterus, another restriction digestion reaction with single restriction endonuclease Mae II was needed. The semi-nested PCR-RFLP was demonstrated to be reliable in species identification of salted fish products in this study. (c) 2005 Elsevier Ltd. All rights reserved.

Identification and analysis of a Scophthalmus maximus ferritin that is regulated at transcription level by oxidative stress and bacterial infection

Ferritins are conserved Iron storage proteins that exist in most living organisms and play an essential role in Iron homeostasis. In this study, we reported the identification and analysis a ferritin M subunit, SmFerM, from turbot Scophthalmus maximus. The full length cDNA of SmFerM contains a 5'-untranslated region (UTR) of 232 bp, an open reading frame (ORF) of 531 bp, and a 3'-UTR of 196 bp The ORF encodes a putative protein of 176 amino acids, which shares extensive sequence identities with the M terrains of several fish species. In silico analysis identified in SmFerM both the ferroxidase center of mammalian H ferritins and the iron nucleation site of mammalian L ferritins. Quantitative real time reverse transcriptase-PCR analysis indicated that SmFerM expression was highest in muscle and lowest in heart and responded positively to experimental challenges with bacterial pathogens and poly(I center dot C) Exposure of cultured turbot hepatocytes to treatment of stress inducers (iron, copper, and H2O2) significantly upregulated the expression of SmFerM in a dose dependent manner. Iron chelating analysis showed that recombinant SmFerM purified from Escherichia coli exhibited apparent iron binding activity. These results suggest that SmFerM is a functional M ferritin and is likely to play a role in iron sequestration and protection against oxidative stress and microbial infection (C) 2010 Elsevier Inc All rights reserved