Identification and expression analysis of terminal deoxynucleotidyl transferase in humphead snapper Lutjanus sanguineus.

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.

Molecular cloning and characterization of lymphocyte cell kinase from humphead snapper (Lutjanus sanguineus).

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 LsLCK). Sequence analysis showed that the full-length cDNA of LsLCK 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 LsLCK 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 LsLCK protein localized distinctly in cytoplasm of cell in thymus, spleen and head kidney. Taken together, these findings indicated that LsLCK may play an important role in the immune response of humphead snapper against bacterial infection.

Cloning, expression of Vibrio alginolyticus outer membrane protein-OmpU gene and its potential application as vaccine in crimson snapper, Lutjanus erythropterus Bloch.

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-OmpU was cloned and expressed in Escherichia coli. Polyclonal antibodies were raised in rabbits against the purified recombinant OmpU, and the reaction of the antibody was confirmed by Western blotting using the isolated OmpU and the recombinant OmpU of V. alginolyticus. To analyze the immunogenicity of the recombinant OmpU, 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 OmpU 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 OmpU is an effective vaccine candidate against V. alginolyticus in L. erythropterus.

Long PCR-RFLP of 16S-ITS-23S rRNA genes: a high-resolution molecular tool for bacterial genotyping.

AIMS: To perform a systematic evaluation of the applicability, validity and reliability of the long PCR-RFLP of 16S-ITS-23S rRNA genes for bacterial genotyping using both sequences retrieved from public genome databases and the experimental data obtained on bacterial cultures. METHODS AND RESULTS: 3301 Full-length sequences of 16S-ITS-23S rRNA genes were retrieved from 885 published bacterial genomes. Copy numbers of the whole set of 16S-ITS-23S rRNA genes per genome ranged from 1 (n = 161) to 14 (n = 4) with an average of 3.71. Their length varied greatly, from 4319 to 6568 bp with an average of 4952 bp. Computer-simulated RFLP analyses of the 16S-ITS-23S fragments flanked by the conserved primers 27F and 2241R suggested MspI, RsaI, HhaI and TaqI as the most appropriate enzymes for long PCR-RFLP analysis of the 16S-ITS-23S sequence. MspI was used to screen over 900 bacterial cultures isolated from the Huguangyan Maar Lake in southern China. An experimental sequencing of 16S rRNA genes of the isolates possessing a unique RFLP band pattern proved the broad applicability and high resolution of this approach. CONCLUSIONS: These results indicate that long PCR-RFLP of 16S-ITS-23S rRNA genes is a potentially universal and reliable bacterial genotyping tool with a high resolution. SIGNIFICANCE AND IMPACT OF THE STUDY: The methodology of long PCR-RFLP of 16S-ITS-23S rRNA genes will facilitate the exploration and tracing of cultivable microbial diversity in natural environments.

Construction of a fusion flagellin complex and evaluation of the protective immunity of it in red snapper (Lutjanus sanguineus).

AIMS: The main aims of this study were to construct a bivalent subunit vaccine containing flagellin flaA gene and flagellin flaB gene from Vibrio alginolyticus strain HY9901 and to explore the potential application of the fusion protein FlaA-(G(4) S)(3)-FlaB as a vaccine candidate for red snapper (Lutjanus sanguineus). METHODS AND RESULTS: Flagellin gene flaA and flaB of V. alginolyticus were linked by gene SOEing (gene splicing by overlap extension) technology. The expression of the fusion gene flaA-(G(4)S)(3)-flaB in Escherichia coli BL21(DE3) was confirmed by SDS-PAGE. Western blot analysis showed that the fusion protein FlaA-(G(4)S)(3)-FlaB, which was purified by affinity chromatography on Ni-NTA resin, had positive reaction with mouse anti-FlaA serum and mouse anti-FlaB serum, respectively. The immunoprotection of FlaA-(G(4) S)(3)-FlaB as a bivalent subunit vaccine was investigated in red snapper model by enzyme-linked immunosorbent assay (ELISA) and challenge test. Red snapper vaccinated with FlaA-(G(4) S)(3)-FlaB produced specific antibodies and were highly resistant to infection by virulent V. alginolyticus. CONCLUSIONS: The fusion gene flaA-(G(4) S)(3)-flaB from V. alginolyticus strain HY9901 was cloned by gene SOEing and was expressed in E. coli. This fusion protein FlaA-(G(4) S)(3)-FlaB is a good protective antigen of V. alginolyticus and should be considered as an effective vaccine candidate against infection by V. alginolyticus in red snapper. SIGNIFICANCE AND IMPACT OF THE STUDY: Two flagellin genes, flaA and flaB, which are independent in structure and function, were first linked together by gene SOEing technology. The finding that red snapper did adequately respond to the fusion protein FlaA-(G(4) S)(3) -FlaB injection made it a promising candidate for vaccine treatment. To develop effective vaccine candidates against V. alginolyticus, more attention should be given to these immunogenic flagellins.

Cloning and expression analysis of recombination activating genes (RAG1/2) in red snapper (Lutjanus sanguineus).

Recombination activating genes (RAG1 and RAG2), involved in the V(D)J recombination of immunoglobulin and T-cell receptor genes play a crucial role in the adaptive immune response in vertebrates. The expression of these genes was required for the proper development and maturity of lymphocytes so that they can be used as useful markers to evaluate the development of lymphoid organ. In this paper, the cDNA of RAG1 and RAG2 in red snapper, Lutjanus sanguineus were cloned by homological cloning and rapid amplification of cDNA ends (RACE) methods. Results showed the full length of RAG1 cDNA was 3944 bp, containing a 5' untranslated region (UTR) of 200 bp, a 3'-UTR of 561 bp and an open reading frame of 3183 bp encoding 1060 amino acids. Three important structural motifs, a RING/U-box domain, a RING/FYVE/PHD-type domain and a RAG Nonamer-binding domain were detected in the deduced amino acid sequence of RAG1 by InterProScan analysis. The full length of RAG2 cDNA was 2200 bp, consisting of a 141 bp 5'-UTR, a 457 bp 3'-UTR and an open reading frame of 1602 bp encoding 533 amino acids. Two important structural motifs, a Galactose oxidase/kelch, beta-propeller domain and a kelch-type beta-propeller domain were detected in the deduced amino acid sequence of RAG2 by InterProScan analysis. BLAST analysis revealed that the RAG1 and RAG2 in red snapper shared a high homology with other known RAG1 and RAG2 genes, while the greatest degree of identity was observed with Hippoglossus hippoglossus RAG1 at 82% and Takifugu rubripes RAG2 at 87%, respectively. The differential expressions of RAG1 and RAG2 in various tissues of red snapper were analyzed by fluorescent quantitative real-time PCR. The overall expression pattern of the two genes was quite similar. In healthy red snappers, the RAGs transcripts were mainly detected in thymus, following head kidney, spleen, intestine, liver and brain. After vaccinated with inactivated Vibrio alginolyticus 48 h later, the RAGs mRNA expression was significantly up-regulated in all studied tissues of red snapper. A clear time-dependent expression pattern of RAG1 and RAG2 after immunization and the expression reached the highest level at 48 h in thymus, 60 h in head kidney and spleen, respectively. These findings indicated that RAG1 and RAG2 could play an important role in the immune response to bacteria in red snapper.

Expression pattern of heat-shock cognate 70 gene of humphead snapper, Lutjanus sanguineus (Cuvier), infected by Vibrio harveyi.

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.

Expression pattern of heat shock protein 90 gene of humphead snapper Lutjanus sanguineus during pathogenic Vibrio harveyi stress.

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.

Protection of red snapper (Lutjanus sanguineus) against Vibrio alginolyticus with a DNA vaccine containing flagellin flaA gene.

AIMS: The main aims of this study were to construct a DNA vaccine containing flagellin flaA gene from Vibrio alginolyticus strain HY9901 and to explore the potential application of pcDNA-flaA as a DNA vaccine candidate for red snapper (Lutjanus sanguineus). METHODS AND RESULTS: Plasmid DNA encoding flagellin flaA gene (designated as pcDNA-flaA) was used as a DNA vaccine to immunize red snapper. The distribution, expression and immunoprotection of the DNA vaccine were analysed in tissues of the red snapper by PCR, RT-PCR and challenge test. PCR results indicated that pcDNA-flaA distributed in liver, spleen, kidney, gill and injection site muscle at 7-28 days after vaccination. RT-PCR results indicated that the flaA gene was expressed in all above tissues of vaccinated fish at 7-28 days after vaccination. In addition, fish receiving the DNA vaccine developed a protective response to live V. alginolyticus challenge 28 days post inoculation, the relative per cent survival (RPS) was 88%. CONCLUSIONS: This study showed that injection of pcDNA-flaA induced an efficient, systemic and antigen-specific immune response in red snapper, which makes it an effective vaccine candidate against V. alginolyticus infection. SIGNIFICANCE AND IMPACT OF THE STUDY: The finding that red snapper does adequately respond to pcDNA-flaA intramuscular injection makes pcDNA-flaA a promising candidate for DNA vaccine treatment. Furthermore, the availability of red snapper for foreign gene expression represents a useful model to develop effective prophylactic strategies and opens new perspectives for the treatment of bacterial pathogens of marine cultured fish.

Immunoproteomic analysis and identification of novel immunogenic proteins from Vibrio harveyi.

AIMS: The main aim of this study was to screen novel immunogenic proteins of Vibrio harveyi, which could be vaccine candidates. METHODS AND RESULTS: Whole-cell proteins of V. harveyi, strain Li01 and Huang01, were first separated by isoelectric focusing, followed by 2D-PAGE, respectively. Immunogenic proteins were identified by Western blotting, using Epinephelus coioides antisera against V. harveyi strain Li01. Western blot analyses revealed 16 shared immunogenic protein spots in both strains. All of the immunogenic proteins were successfully identified and corresponded to 15 proteins. None of these proteins have been previously reported as immunogenic for V. harveyi. Of the 15 proteins, 11 are specific immunoreactive proteins and four are nonspecific immunoreactive proteins. Furthermore, outer membrane protein N (spot 2) and oligopeptide ATP-binding cassette (ABC) transporter (spot 3) were used as immunogens to immunize E. coioides for investigation of their protective abilities and activities. The E. coioides immunized with OmpN has abilities to fight against infections caused by V. harveyi Li01 and Huang01. However, vaccination with oligopeptide ABC transporter induces low protective immune response in fish. CONCLUSIONS: Eleven novel specific antigens were found, and OmpN could potentially be used as vaccine candidate for the development of novel vaccine against V. harveyi. SIGNIFICANCE AND IMPACT OF THE STUDY: These data show that immunoproteomics methods can be successfully applied in identifying immunogenic proteins of V. harveyi, which helps to search for the protective antigens in future.

Evaluation of a loop-mediated isothermal amplification method for the rapid detection of Vibrio harveyi in cultured marine shellfish.

AIMS: The purpose of this study was to establish a loop-mediated isothermal amplification (LAMP) method for the rapid, sensitive detection of Vibrio harveyi in mariculture shellfish. METHODS AND RESULTS: A set of four primers, two outer and two inner primers, were designed from the toxR gene sequence of V. harveyi. The LAMP reaction was conducted at 65 degrees C for 60 min. There were no cross-reactions with other bacterial strains indicating a high specificity of the LAMP. The detection sensitivity of the LAMP assay for V. harveyi with both of pure cultures and added shellfish cultures is about 10(-5) dilution level (equivalent to 17.2 cells per reaction). The amplification products were detected by visual inspection using SYBR Green I. The detection sensitivity using the LAMP method was 10 times higher than that of conventional PCR. CONCLUSIONS: The LAMP assay established in this study is an extremely specific, sensitive and rapid for identification of V. harveyi in mariculture shellfish. SIGNIFICANCE AND IMPACT OF THE STUDY: This LAMP technique provides an important detecting tool for the detection of V. harveyi infection both in the laboratory and field. This technique is recommended as an applied protocol for health management programme and disease surveillance of in hatcheries as well as in grow-out pond, to prevent the disease outbreak.

Loop-mediated isothermal amplification method for rapid detection of Vibrio alginolyticus, the causative agent of vibriosis in mariculture fish.

AIMS: The purpose of this study was to develop a loop-mediated isothermal amplification (LAMP) method for the rapid, sensitive and simple detection of Vibrio alginolyticus in mariculture fish. METHODS AND RESULTS: LAMP primers were designed by targeting the gyrB gene. With Bst DNA polymerase, the target DNA can be clearly amplified for 60 min at 64 degrees C in a simple water bath. The detection sensitivity of the LAMP assay for the detection of V. alginolyticus is about 3.7 x 10(2) CFU ml(-1) (3.7 CFU per reaction). LAMP products could be judged with agar gel or naked eye after the addition of SYBR Green I. There were no cross-reactions with other bacterial strains indicating a high specificity of the LAMP. The LAMP method was applied to detect V. alginolyticus-infected fish tissues effectively. CONCLUSIONS: The LAMP established in this study is a simple, sensitive, specific, inexpensive and rapid protocol for the detection of V. alginolyticus. SIGNIFICANCE AND IMPACT OF THE STUDY: This LAMP method provides an important diagnostic tool for the detection of V. alginolyticus infection both in the laboratory and field.

Expression, purification and antibody preparation of flagellin FlaA from Vibrio alginolyticus strain HY9901.

AIMS: The main aims of this study were to clone and express flagellin flaA gene from Vibrio alginolyticus strain HY9901, also to prepare mouse anti-FlaA polyclonal antibody for future pathogen or vaccine study. METHODS AND RESULTS: The full-length flaA gene was amplified by PCR with designed primers. The open reading frame of flaA gene contains 1131 bp, and its putative protein consists of 376 amino acid residues. Alignment analysis indicated that the FlaA protein was highly conserved. SDS-PAGE indicated that the FlaA protein was successfully expressed in Escherichia coli BL21 (DE3). Then, the recombinant FlaA protein was purified by affinity chromatography, and the mouse anti-FlaA serum was produced. The expression of flaA gene was verified by various immunological methods, including western blotting, enzyme-linked immunosorbent assay (ELISA) and immunogold electron microscopy (IEM). CONCLUSIONS: Flagellin flaA gene was cloned and identified from V. alginolyticus HY9901, the recombinant FlaA protein was expressed and purified, and high-titre FlaA protein-specific antibody was produced. Western blot analysis revealed that the prepared antiserum not only specifically react to FlaA fusion protein, but also to natural FlaA protein of V. alginolyticus. The expressed FlaA protein was demonstrated, for the first time, as the component of flagella from V. alginolyticus by IEM. SIGNIFICANCE AND IMPACT OF THE STUDY: This study may offer important insights into the pathogenesis of V. alginolyticus, provide a base for further studies on the diagnosis and evaluation that whether the FlaA protein could be used as an effective vaccine candidate against infection by V. alginolyticus and other Vibrio species. Additionally, the purified FlaA protein and polyclonal antibody can be used for further functional and structural studies.

Cloning and expression of the gene encoding an extracellular alkaline serine protease from Vibrio alginolyticus strain HY9901, the causative agent of vibriosis in Lutjanus erythopterus (Bloch).

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 LD(50) of 0.25 microg protein g(-1) body weight.

Cloning and expression of gene encoding the thermostable direct hemolysin from Vibrio alginolyticus strain HY9901, the causative agent of vibriosis of crimson snapper (Lutjanus erythopterus).

AIMS: The main aims of this study were to clone and express complete open reading frame (ORF) of thermostable direct haemolysin gene (tdh) from Vibrio alginolyticus strain HY9901 in Escherichia coli, and further evaluate the virulence of expressed TDH on mouse and crimson snapper. METHODS AND RESULTS: A 410 bp internal fragment of the tdh gene was amplified by touchdown PCR with designed primers. Then its unknown flanking sequences of the 5'- and 3'-ends were finally characterized by inverse PCR and nested PCR. Sequence analysis showed that the tdh gene contain 570 bp ORF which encoded 189 amino acids. The deduced amino acid sequence of the ORF was in significant homology with several Vibrio TDH. The product that the tdh gene expressed in E. coli was purified by Ni(2+)-IDA Sepharose affinity column. The activity of purified TDH was 4651 U mg(-1) protein by hide powder azure digestion. The lethal toxicity test showed that LD(50) values of the purified TDH were 5.68 and 8.34 microg TDH g(-1) body weight for mouse and crimson snapper, respectively. CONCLUSIONS: The complete ORF of tdh gene was obtained by touchdown PCR, inverse PCR and nested PCR. The ORF was perfectly expressed in E. coli. The activity and toxicity assays showed that the N-terminal signal peptide was essential to autocatalyse and fold correctly to obtain the activity and toxicity in the purified TDH. The Native-PAGE analysis showed that the activated tdh gene expressed in E. coli was a dimer with two identical subunits. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that the expressed activated TDH can produce the toxicity protein determined on mouse and fish, which will lead to better understandings of the identifying virulence factor that could be considered as a candidate antigen for vaccine and a diagnostic tool for vibriosis. Its use as an immunizing antigen might prevent the ability of V. alginolyticus to infect the marine aquatic animals, as a complementary measure to tick control and appropriate management in countries affected by vibriosis.