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A tdt gene was identified successfully from humphead snapper Lutjanus sanguineus, which contained 1710 bp encoding a protein of 463 amino acids. Results of quantitative real-time polymerase chain reaction ( qRT-PCR) indicated that tdt mainly expressed in thymus and head kidney and the transcripts of tdt in these tissues were up-regulated significantly at 36 and 48 h after Vibrio harveyi infection. Meanwhile Tdt-producing cells were found in thymus and head kidney. [ABSTRACT FROM AUTHOR]

Lymphocyte cell kinase ( LCK) belongs to the Src family of tyrosine kinases, which involves in the proliferation control of lymphocytes. In this study, we cloned the LCK gene of humphead snapper ( Lutjanus sanguineus) (designed as Ls LCK). Sequence analysis showed that the full-length cDNA of Ls LCK was 2279 bp, contained a 1506-bp open reading frame ( ORF), encoding a polypeptide of 501 amino acids. The deduced amino acid possessed the typical structural features of known LCK proteins, including four Src homology ( SH) domains arranged as the SH1 domain followed by a regulatory C-terminal tail ( COOH-domain), SH2 and SH3 adapter domains and SH4 domain which required for membrane attachment and CD4/ CD8 binding. Fluorescent quantitative real-time PCR analysis indicated that Ls LCK transcripts were expressed mainly in thymus, spleen and head kidney in healthy fish. Moreover, the mRNA expressions in these tissues were significantly up-regulated after challenge with Vibrio harveyi. The results of immunohistochemistry showed that Ls LCK protein localized distinctly in cytoplasm of cell in thymus, spleen and head kidney. Taken together, these findings indicated that Ls LCK may play an important role in the immune response of humphead snapper against bacterial infection. [ABSTRACT FROM AUTHOR]

The outer membrane proteins of the marine aquatic animal pathogen, Vibrio alginolyticus, play an important role in the virulence of the bacterium and are potential candidates for vaccine development. In this study, the gene encoding an outer membrane protein- Omp U was cloned and expressed in Escherichia coli. Polyclonal antibodies were raised in rabbits against the purified recombinant Omp U, and the reaction of the antibody was confirmed by Western blotting using the isolated Omp U and the recombinant Omp U of V. alginolyticus. To analyze the immunogenicity of the recombinant Omp U, crimson snapper, Lutjanus erythropterus Bloch, were immunized by intraperitoneal injection, and antibody response was assessed by the enzyme-linked immunosorbent assay ( ELISA). The results demonstrated that the recombinant Omp U produced an observable antibody response in all sera of the vaccinated fish. The vaccinated fish were challenged by virulent V. alginolyticus and observed to have high resistance to infection. These results indicate that the recombinant Omp U is an effective vaccine candidate against V. alginolyticus in L. erythropterus. [ABSTRACT FROM AUTHOR]

The purpose of this study was to develop a loop-mediated isothermal amplification (LAMP) method for rapid, sensitive, and simple detection of Streptococcus agalactiae in farmed fish. A set of four primers, two outer and two inner, was designed from the fibrinogen-binding (fbsB) proteins gene sequence of S. agalactiae, and conditions for LAMP were optimized as incubation of all reagents for 60 min at 64°C. LAMP products were judged by agar gel electrophoresis as well as the naked eye after development by Synergy Brands Green I (SYBR Green I). Sensitivity of the assay was 2.8 x 103 CFU/ml (2.8 CFU per reaction) and no positive amplification was detected for other related bacteria. This LAMP method can be used to detect S. agalactiae infection in the laboratory and the field. [ABSTRACT FROM AUTHOR]

The heat-shock cognate 70 (HSC70) gene of humphead snapper, Lutjanus sanguineus, designated as ByHSC70, was cloned by rapid amplification of cDNA ends (RACE) with the primers designed from the known expressed sequence tag (EST) identified from the subtracted cDNA library of the head kidney of humphead snapper. The full-length cDNA of ByHSC70 is 2313 bp, containing a 5′ terminal untranslated region (UTR) of 96 bp, a 3′ terminal UTR of 267 bp, and an open reading frame (ORF) of 1950 bp encoding a polypeptide of 650 amino acids with a theoretical molecular weight of 71.21 kDa and an estimated isoelectric point ( pI) of 5.08. ByHSC70 contained three classical HSP70 family signatures. BLAST analysis showed that the amino acid sequence of ByHSC70 had the highest similarity of 99% when compared with other HSC70s. Fluorescent real-time quantitative RT-PCR was used to examine the expression of ByHSC70 gene in eight kinds of tissues/organs of humphead snapper after challenge with Vibrio harveyi. There was a clear time-dependent expression pattern of ByHSC70 in head kidney, spleen and thymus after bacterial challenge, and the expression of mRNA reached a maximum level at 9, 6 and 24 h post-infection and then returned to control levels after 15, 24 and 36 h, respectively. Our results suggest that HSC70 is an important component in the immune system of humphead snapper, its their rapid transcriptional upregulation in response to V. harveyi infection might be important for survival of humphead snapper. [ABSTRACT FROM AUTHOR]

The full-length cDNA of heat shock protein 90 (HSP90) of humphead snapper Lutjanus sanguineus, designated as rsHSP90, was cloned by rapid amplification of complementary (c)DNA ends (RACE) techniques with the primers designed from the known expressed sequence tag (EST) sequence identified from the subtracted cDNA library of the head kidney of L. sanguineus. Sequence analysis showed that the full-length cDNA of rsHSP90 was 2745 bp, containing a 5′ terminal untranslated region (UTR) of 99 bp, a 3′ terminal UTR of 471 bp and an open reading frame (ORF) of 2175 bp encoding a polypeptide of 725 amino acids. On the basis of the deduced amino acid sequence, the theoretical molecular mass of rsHSP90 was calculated to be 83·18 kDa with an isoelectric point of 4·79. Moreover, five classical HSP90 family signatures were found in the amino acids sequence of rsHSP90 by PredictProtein. Basic local-alignment search-tool (BLAST) analysis revealed that the amino acids sequence of rsHSP90 had the highest similarity of 97% when compared with other HSP90s. Fluorescent real-time quantitative reverse-transcription (RT)-PCR was used to examine the expression pattern of rsHSP90 in eight kinds of tissues and organs of L. sanguineus challenged with Vibrio harveyi. There was a clear time-dependent expression pattern of rsHSP90 in head kidney, spleen and thymus after bacterial challenge and the expression of messenger (m)RNA reached the maximum level at the time points of 9, 15 and 24 h, respectively. The up-regulated mRNA expression of rsHSP90 in L. sanguineus after bacterial challenge indicated that rsHSP90 was inducible and might be involved in immune response. [ABSTRACT FROM AUTHOR]

A 750-bp internal fragment of the alkaline serine protease gene ( asp) from the Vibrio alginolyticus strain HY9901 was amplified by polymerase chain reaction (PCR). The flanking sequences of the 5′- and 3′- ends of the asp gene were characterized by reverse and nested PCR. Sequence analysis showed that the asp gene contained an 1893-bp ORF encoding 630 amino acids. The deduced amino acid sequence of the ASP (alkaline serine protease) precursor showed significant homology with several bacterial alkaline serine proteases. Expression of the asp gene in Escherichia coli and activity tests of the ASP indicated that the N-signal peptide of the ASP precursor was essential to autocatalyse and fold correctly the enzyme to obtain activity. The purified ASP was lethal for Lutjanus erythopterus with an LD50 of 0.25 μg protein g−1 body weight. [ABSTRACT FROM AUTHOR]