[Objectives] To analyze the function of cobQ gene from Vibrio alginolyticus strain HY9901,and to provide a reference for exploring the possible mechanism of cobQ gene from V.alginolyticus.[Methods] A pair of primers w...[Objectives] To analyze the function of cobQ gene from Vibrio alginolyticus strain HY9901,and to provide a reference for exploring the possible mechanism of cobQ gene from V.alginolyticus.[Methods] A pair of primers were designed based on the sequence of the V.alginolyticus cobQ gene and used to amplify the full-length gene by PCR.[Results] The PCR amplification results indicated that the cobQ gene has a full length of 780 bp,encoding 259 amino acid residues.The deduced amino acid sequence predicts a molecular weight of approximately 28.83 kD and an isoelectric point of 9.21.Sequence analysis revealed no N-terminal signal peptide cleavage site,suggesting the absence of both a signal peptide and transmembrane regions in this protein.The amino acid sequence contains 2 N-terminal myristoylation sites,1 N-glycosylation site,1 glycosaminoglycan attachment site,4 microbody C-terminal targeting signal sites,3 casein kinase II phosphorylation sites,and 4 protein kinase C phosphorylation sites.Subcellular localization prediction showed that the CobQ protein is primarily localized in the cytoplasm(65.2%probability).Homology analysis demonstrated that the amino acid sequence of the cobQ gene from V.alginolyticus shares up to 99%homology with other Vibrio species,clustering within the same subclade as Vibrio parahaemolyticus,indicating close phylogenetic relationships.Secondary structure prediction revealed proportions ofα-helices,random coils,and extended strands as 44.40%,36.68%,and 18.92%,respectively.The tertiary structure model exhibited 87.62%similarity to the template A0A165XBE1.1.[Conclusions] In this study,the V.alginolyticus cobq gene was successfully cloned and its sequence was analyzed by bioinformatics.It is expected to lay a foundation for the subsequent study of the regulatory mechanism of its protein on the virulence of V.alginolyticus.展开更多
[Objectives]This study was conducted to investigate the functional characteristics of the trxB gene in Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the trxB gene sequence of V.alginol...[Objectives]This study was conducted to investigate the functional characteristics of the trxB gene in Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the trxB gene sequence of V.alginolyticus for PCR cloning of its full-length sequence.Systematic bioinformatics analyses were conducted to predict the physicochemical properties,secondary structure,and tertiary structure of the encoded protein.[Results]The trxB gene is 960 bp in length,encoding 319 amino acid residues.The deduced protein has a predicted molecular weight of 34.32 kDa and an isoelectric point(pI)of 4.77.Analysis of the amino acid sequence revealed a distinct signal peptide cleavage site at the N-terminus,with no transmembrane domains.The functional sites are as follows:1 N-glycosylation site,1 cAMP-and cGMP-dependent protein kinase phosphorylation site,4 protein kinase C phosphorylation sites,7 casein kinase II phosphorylation sites,1 tyrosine kinase phosphorylation site,11 N-myristoylation sites,1 prenyl group binding site,3 microbody C-terminal targeting signal sites,and 1 xanthine nucleotide-disulfide oxidoreductase class II active site.Subcellular localization prediction indicated the highest probability(44.4%)for endoplasmic reticulum localization.The TrxB amino acid sequence of V.alginolyticus shares 97.2%-98.4%homology with other Vibrio species,and they were clustered within the same subgroup.Secondary structure prediction showed proportions of random coils(31.97%),alpha-helices(31.66%),extended strands(25.08%),and beta turns(11.29%).The tertiary structure model exhibited 88.68%similarity to template 5vt3.1.A.[Conclusions]This study elucidated the characterization of the TrxB protein in V.alginolyticus,laying a theoretical foundation for the development of outer membrane protein subunit vaccines against this pathogen.展开更多
[Objectives]To develop a pair of specific primers for the PCR amplification of the full-length relA gene from Vibrio alginolyticus strain HY9901,as well as to conduct bioinformatics analysis.[Methods]The relA gene was...[Objectives]To develop a pair of specific primers for the PCR amplification of the full-length relA gene from Vibrio alginolyticus strain HY9901,as well as to conduct bioinformatics analysis.[Methods]The relA gene was amplified through PCR,and the resulting gene sequence was subsequently analyzed using bioinformatics tools,including amino acid sequence prediction,functional site analysis,subcellular localization prediction,and homology comparison.[Results]The relA gene had a total length of 2220 bp and encoded 739 amino acid residues.The molecular weight was approximately 84.1261 kDa,and its isoelectric point was 5.95.The protein lacked a signal peptide and transmembrane regions,while exhibiting multiple phosphorylation sites.Predictions regarding its subcellular localization suggested that it was predominantly situated in the cytoplasm.The amino acid sequence demonstrated a homology of 97%to 99%with other species within the genus Vibrio,and it clustered within the same subfamily as V.antiquarius and V.diabolicus.In the prediction of secondary structure,the proportions ofα-helix,extended strand,random coil,andβ-sheet were 54.13%,12.04%,28.15%and 5.68%,respectively.The similarity between the tertiary structure model and template 5kpw.1.w was 66%.[Conclusions]In this study,the relA gene of V.alginolyticus strain HY9901 has been successfully amplified and analyzed.The structural characteristics and potential functions of the encoded protein have been elucidated,thereby providing foundational data for understanding the role of this gene in V.alginolyticus.展开更多
[Objective] The purpose of this study was to construct a vscC in-frame deletion mutant of Vibrio alginolyticus with no antibiotic resistance marker. [Method] The first vscC mutant molecules in vitro were generated by ...[Objective] The purpose of this study was to construct a vscC in-frame deletion mutant of Vibrio alginolyticus with no antibiotic resistance marker. [Method] The first vscC mutant molecules in vitro were generated by SOE-PCR and then lig- ated to a suicide vector pDM4 to construct a suicide recombinant vector pDM4- A vscC. To clone the recombinant vector, it was transformed to E. coli SY327 strain, and then positive clones were selected and proved by PCR analysis. After that, the pDM4-AvscC DNA was extracted in large numbers and transformed to the E. coil S17-1 strain that acted as a donor in bacterial conjugation using the heat shock method. The recombinant E. coli S17-1 strains then transferred the pDM4-AvscC to V. alginolyticus ZJ51-O by conjugation method; transconjugants were screened and selected sequentially using antibiotic selection strategy and sucrose based counter-selection system to find the suspected mutants wanted. Finally the putative mutants were identified by PCR and confirmed by sequencing analysis. [Result] ZJ51-OAvscC was successfully constructed. [Conclusion] This study laid foundation for further research on the function of vscC gene and molecular mechanism of type Ⅲ secretion system in V. alginolyticus. Simultaneously, by the effective method other unknown functional genes in V. alginolyticus genome would be researched.展开更多
Interleukin 1β（IL-1β）, the first interleukin to be characterized, plays a key role in regulating the immune response. In this study, we determined the c DNA and genomic DNA sequences of the IL-1β gene from the la...Interleukin 1β（IL-1β）, the first interleukin to be characterized, plays a key role in regulating the immune response. In this study, we determined the c DNA and genomic DNA sequences of the IL-1β gene from the large yellow croaker, Larimichthys crocea. Phylogenetic analysis indicated that the IL-1β（Lc IL-1β） gene was most closely related to that of European seabass（Dicentrarchus labrax）, sharing 67.8% amino acid identity. In healthy large yellow croaker, Lc IL-1β transcription was detected in all tested tissues, with the highest level found in the head kidney. Upon Vibrio alginolyticus infection, Lc IL-1β transcription in all tested tissues was significantly upregulated. Intraperitoneal injection of recombinant Lc IL-1β（r Lc IL-1β） improved the survival rate and reduced the tissue bacterial load after V. alginolyticus infection. In addition, r Lc IL-1β induced monocytes/macrophages（MO/MΦ） chemotaxis and increased phagocytosis and bactericidal activity in vitro. These results suggest that Lc IL-1β plays an important role in the large yellow croaker immune response against V. alginolyticus.展开更多
A bstract Gut microbiota impacts the health of crustaceans. V ibrio alginolyticus is a main causative pathogen that induces the vibriosis in farmed swimming crabs, Portunus trituberculatus. However, it remains unknown...A bstract Gut microbiota impacts the health of crustaceans. V ibrio alginolyticus is a main causative pathogen that induces the vibriosis in farmed swimming crabs, Portunus trituberculatus. However, it remains unknown whether gut bacteria perform functions during the progression of vibriosis. In this study, 16 SrRNA gene amplicon sequencing was used to investigate temporal alteration of gut bacterial community in swimming crabs in response to 72-h V. alginolyticus challenge. Our results show that V. alginolyticus infection resulted in dynamic changes of bacterial community composition in swimming crabs. Such changes were highlighted by the overwhelming overabundance of V ibrio and a significant fluctuation in the gut bacteria including the bacteria with high relative abundance and especially those with low relative abundance. These findings reveal that crab vibriosis gradually develops with the infection time of V. alginolyticus and tightly relates to the dysbiosis of gut bacterial community structure. This work contributes to our appreciation of the importance of the balance of gut bacterial community structure in maintaining the health of crustaceans.展开更多
Adhesion of Vibrio alginolyticus to the gill mucus of Pseudosciaena crocea has been investigated using [ methyl-^3 H ] thymidine as isotope tracer. The results showed that： the adhesive quantity of V. alginolyticus i...Adhesion of Vibrio alginolyticus to the gill mucus of Pseudosciaena crocea has been investigated using [ methyl-^3 H ] thymidine as isotope tracer. The results showed that： the adhesive quantity of V. alginolyticus increased with bacterial concentrations and reached equilibrium after incubated for 180 min; the higher adhesive quantity was obtained at 15 ~ 30 ℃ and sourish conditions; adhesion of V. alginolyticus could not achieved without Na^＋ , and Ca^2＋ played an auxiliary role in the bacterial adhesion; adhesion of V. alginolyticus was inhibited remarkably by starvation, heat treatment and periodic acid treatment; all of the eight kinds of carbohydrates investigated enhanced the adhesion of V. alginolyticus to the gill mucus of P. crocea, among them, glucose, mannose, fructose and maltose showed the specially enhanced adhesion. The results indicated that E alginolyticus could adhere to the gill mucus of P. crocea facilely in seawater, and this bacterial adhesion was influenced by environmental factors and closely related to superficial carbohydrate structures and some heat-sensitive structures.展开更多
For the investigation of anti-infection immune response of Pseudosciaena crocea, 160 healthy fish samples were categorized into infected and control groups. Each individual fish in the infected group was injected intr...For the investigation of anti-infection immune response of Pseudosciaena crocea, 160 healthy fish samples were categorized into infected and control groups. Each individual fish in the infected group was injected intraperitoneally (i.p.) with 0.2 ml bacterial suspension of Vibrio alginolyticus in density of 2×107 CFU/ml, while each individual in the control group was injected i.p. with 0.2 ml sterile saline solution (0.85%). It was observed that the artificial injection of V. alginolyticus significantly increased the number of erythrocytes, leucocytes, lymphocytes in peripheral blood as well as peripheral serum antibacterial activity and antibody titer of large yellow croaker, and significantly reduced the number of peripheral blood granulocytes as compared with those in the control group. No significant difference in acid phosphytase and superoxide dismutase activity of serum was detected between the two groups. It is suggested that non-specific immune factors including leucocytes and anti-bacteria substance in peripheral blood played important role at the initial stage of infection, and specific immune factors such as antibody then played important role in response to anti-infection at the latter stage.展开更多
[Objectives]The purpose was to investigate the function and mechanism of exsA gene in type III secretion system of Vibrio alginolyticus.[Methods]The full length of exsA was cloned using molecular biology techniques to...[Objectives]The purpose was to investigate the function and mechanism of exsA gene in type III secretion system of Vibrio alginolyticus.[Methods]The full length of exsA was cloned using molecular biology techniques to analyze its biological information.Fluorescence quantitative PCR technology was used to analyze the expression of exsA after different media stress.[Results]The exsA gene contains an open reading frame(ORF)of 861 bp,encoding 286 amino acids.The physico-chemical analysis shows that the molecular structural formula is C1442H2267N393O441S12,the theoretical molecular weight is 32.549 kD,the theoretical pI value is 6.0,and the protein is non-hydrophilic and unstable.The gene does not contain a transmembrane region,and there is no obvious signal peptide.The prediction result of protein subcellular localization shows that the protein is inside the cell.The deduced amino acid sequence and constructed phylogenetic tree show that V.alginolyticus has a close relationship with Vibrio antiquarius.The qPCR results show that the expression level of exsA in different media is different,highest in TSB medium containing bile salts,followed by DMEM medium,and lowest in ordinary TSB medium.[Conclusions]The gene sequence,molecular structure and isoelectric point of exsA,as well as its expression in three different media were obtained.展开更多
In this study, Va 1686 gene was cloned from Vibrio alginolyticus . The total length of the gene is 1 164 bp, and it could encode 387 amino acids. The physicochemical properties, protein structure, genetic evolutionary...In this study, Va 1686 gene was cloned from Vibrio alginolyticus . The total length of the gene is 1 164 bp, and it could encode 387 amino acids. The physicochemical properties, protein structure, genetic evolutionary relationship and antigenic characteristics of the effect protein Va1686 of V. alginolyticus HY9901 type Ⅲ secretion system were studied and analyzed by bioinformatics methods and tools. The results showed that Va1686 is a stable hydrophilic and acidic protein without a transmembrane region and a signal peptide, and secondary structure to α-helix. The evolutionary analysis showed that V. alginolyticus HY9901 and V. harveyi were clustered together, which indicated that the genetic relationship between the two species was the closest. Va1686 contains a Fic superfamily conserved domain associated with cell division. Bioinformatics analysis showed that the B-cell preponderant epitopes of Va1686 might be localized in the regions of 48-49, 82-85, 125-126, 150-153, 185-186, 236-237 and so on. The 3D structure model of Va1686 subunit was simulated by SWISS-MODEL software and it was found that the vopS of V. parahaemolyticus was similar and the similarity was 89.46%. In this study, the feasibility of Va1686 as a common antigen of Vibrio was verified from the perspective of bioinformatics, which laid the foundation for the next step in vaccine development.展开更多
In this paper, the hopPmaJ gene, which encodes type III effector HopPmaJ of Vibrio alginolyticus, was cloned, sequenced and bioinformatically predicted. The results showed that hopPmaJ （GenBank accession number： KX2...In this paper, the hopPmaJ gene, which encodes type III effector HopPmaJ of Vibrio alginolyticus, was cloned, sequenced and bioinformatically predicted. The results showed that hopPmaJ （GenBank accession number： KX245315） is 345 bp in length and encodes 114 amino acids, with a theoretical molecular mass of 12. 774 kD, and a pI of 4.45. The protein has multiple functional domains and binding sites, but no signal peptide. The tertiary structure of the protein is a homodimer. Based on multiple parameters, the possible dominant B cell antigenic epitopes of HopPmaJ were predicted to be at positions 13 - 16, 29 -30, 40 -42, 45 -49 and 84 -91.展开更多
[Objectives]To clone and analyze the TpiA gene of Vibrio alginolyticus HY9901.[Methods]According to the TpiA gene sequence of V.alginolyticus,a pair of specific primers was designed,and its full length was amplified b...[Objectives]To clone and analyze the TpiA gene of Vibrio alginolyticus HY9901.[Methods]According to the TpiA gene sequence of V.alginolyticus,a pair of specific primers was designed,and its full length was amplified by PCR.[Results]The full length of TpiA gene is 771 bp,encoding 256 amino acid residues in total,and the NCBI accession number is OM906798.According to the deduced amino acid sequence,its molecular weight was predicted to be about 26.97548 kDa,and its isoelectric point was 4.78.The amino acid sequence of the N-terminal signal peptide structure was predicted,and it was found that there was no obvious signal peptide cleavage site,no signal peptide,and no transmembrane region;the amino acid sequence contained 3 N-glycosylation sites,4 protein kinase C phosphorylation sites,2 casein kinase II phosphorylation sites,6 N-myristoylation sites,7 microbody C-terminal target signal site,and 1 triose phosphate isomerase active site.The prediction results of protein subcellular localization showed that TpiA may be located in mitochondria or cytoplasm,with probability of 39.1%and 34.8%,respectively.The amino acid sequence of the TpiA gene of V.alginolyticus shared 98.83%-99.61%homology with other Vibrio species,and it was clustered into the same subfamily with Vibrio parahaemolyticus and had a close relationship.In the secondary structure prediction,the proportions ofα-helix,random coil and extended chain were 44.53%,41.41%and 14.06%,respectively,and the similarity of its tertiary structure model to template 1aw1.1.A was 85.16%.[Conclusions]This study is intended to provide a basis for further research on the role of TpiA gene in the type III secretion system and related research on antibiotic resistance.展开更多
To get a better understanding of the starvation survival strategy of pathogenic Vibrio alginolyticus, log-phase cells were inoculated into sterile natural seawater for starvation studies. The results showed that all o...To get a better understanding of the starvation survival strategy of pathogenic Vibrio alginolyticus, log-phase cells were inoculated into sterile natural seawater for starvation studies. The results showed that all of total bacteria number, viable bacteria number and CFU number of V. alginolyticus increased remarkably at the initial starvation stage; after reaching their peaks at 5 d, both total bacteria number and viable bacteria number of V. alginolyticus fell slowly, while the CFU number fell more quickly after reaching its peak at 10 d; V. alginolyticus elongated their cells at the prophase of starvation, and then shrunk their volume and turned their shapes into ovals from rods at the anaphase of starvation; starved cells showed more sensitivity to heating and UV; starved cells showed no significant difference from unstarved ones at the lowest detection limit determined by indirect enzyme-linked immu- nosorbent assay （ELISA） ; starved cells＇ ability to adhere to the skin mucus of large yellow croaker （ Pseudosciaena crocea） showed a sharp decline as the starvation time increases; the cellular protein of V. alginolyticus increased remarkably at the ariaphase of starvation. The results indicated that pathogenic V. alginolyticus could survive in starvation for relatively long periods of time （ ≥2 months） in 28℃ natural seawater due to the morphological and physiological changes; however, starved V. alginolyticus cells showed less virulence and higher sensitivity under environmental stresses.展开更多
[ Objeetlve] This study aimed to investigate the role of dihydrolipoamide dehydrogenase （DLDH） in the pathopoiesis of Vibrio alginolyticua. [ Method] Using suicide plasmid pRE112 as the vector, d/dh deletion mutant ...[ Objeetlve] This study aimed to investigate the role of dihydrolipoamide dehydrogenase （DLDH） in the pathopoiesis of Vibrio alginolyticua. [ Method] Using suicide plasmid pRE112 as the vector, d/dh deletion mutant strain ZJO3Adldh was constructed with insertion inactivation mutation method by Overlap PCR and homologous recombination technology to compare and analyze the differences in the growth, swimming ability, enzyme activity, biofilm formation and pathogenicity between wild-type strain ZJ03 and deletion mutant strain ZJO3△dldh. [ Result] dldh deletion mutant strain ZJ03 △d/dh exhibited prolonged lag phase, significantly reduced swimming ability （P 〈 0.05 ）, significantly reduced enzyme activity （P 〈 0.05 ）, significantly reduced biofilm formation ability （P 〈 0.05 ） and remarkably reduced pathogenicity to Epinephelus coioides （P 〈0.05） compared to wild-type strain ZJ03. [ Conclusion] This study provided theoretical basis for revealing the pathogenic mechanisms of Vibrio from the perspective of bacterial energy metabolism.展开更多
Vibrio alginolyticus, a gram-negative bacterium has been described to be one of the most common and economically important aquatic pathogens of fish and shellfish. Vaccine immunization is an effective approach prevent...Vibrio alginolyticus, a gram-negative bacterium has been described to be one of the most common and economically important aquatic pathogens of fish and shellfish. Vaccine immunization is an effective approach preventing V. alginolyticus infection. Attenuated vaccine stimulates systemic immune response in the host, but few attenuated vaccine against V. alginolyticus is available. The type III secretion system （T3SS）, an important pathogenic factor of V. alginolyticus, is used by bacterial pathogens to inject effector proteins into the cytoplasm of their host cells. The T3SS forms a structure called needle complex with a multi-ring base that spans the bacteria and a needle-like extension that protrudes several nanometers from the bacterial surface, vscO locates at the ＂needle＂ site of T3SS, playing the role of escorting the molecular chaperone and effector proteins into host cells and further inducing the death of host cells. In this paper, an in-frame deletion mu- tant of vscO was constructed using overlap PCR and homologous recombination technology combining with chloramphenicol （Cm） and sucrose screening. The LDs0 changes of ZJO3AvscO mutant strains compared with the strain ZJ03 were e^amined in grouper （ Epinephelus coioides）. The ZJO3AvscO mutant showed about 150 times decrease in virulence in E. coioides compared with wide type ZJ03. After vaccination with ZJO3AvscO in E. coioides through injection and immersion, the spe- cific antibody titers were markedly higher than that in the saline control group （P 〈 0.05 ）. The titers of injection and immersion group on the forth week reached the maximums at 1：2 048 and 1： 128, respectively. The relative percentage survival （RPS） of injection group was 84%, while that in immersion group was 68%. These results indicate that the ZJO3AvscO of V. alginolyticus has a high immunogenicity, and can be used as live attenuated vaccine. In addition, RPS may be affected by vaccination and infection methods. This study can provide technical support for controlling fish diseases caused by V. alginolyticus.展开更多
[Objectives]This study aimed to clone the tye A gene of Vibrio alginolyticus HY9901 strain and analyze its sequence by bioinformatics.[Methods] By referring to the entire genome sequence of Ⅴ.alginolyticus on Gen Ban...[Objectives]This study aimed to clone the tye A gene of Vibrio alginolyticus HY9901 strain and analyze its sequence by bioinformatics.[Methods] By referring to the entire genome sequence of Ⅴ.alginolyticus on Gen Bank,specific primers were designed.According to the principle of PCR amplification,the target gene tye A was amplified.By means of bioinformatics,the sequence of tye A was further analyzed,and the phylogenetic tree of tye A genes of Vibrio spp.and the corresponding subunit three-dimensional structure models were constructed.[Results] The length of the tye A gene of Ⅴ.alginolyticus strain HY9901 is 285 bp,and its theoretical molecular weight is 10.98 kD.According to prediction,there is no signal peptide or transmembrane region at the N-terminus of the sequence,and the amino acid sequence contains two casein kinase Ⅱ phosphorylation sites.The results of protein subcellular localization prediction show that the Tye A protein is located in the cell membrane.The protein is unstable and non-hydrophilic.The tertiary structure of Tye A protein of Ⅴ.alginolyticus is similar to that of Yersinia sp.According to prediction,Tye A has a major functional domain Pfam.In terms of secondary structure,alpha helix,random coil,extended strand and beta turn account for 85.11%,7.45%,4.26% and 3.19%,respectively.The homology of Tye A between Ⅴ.alginolyticus and Vibrio parahaemolyticus is up to 83%,so they are classified into one cluster.[Conclusions]This study will help to further understand the regulation mechanism of type Ⅲ secretion system in Ⅴ.alginolyticus.展开更多
The two-component regulatory system plays an important role in the growth and virulence of bacteria. In this study, the phosphate transcriptional regulatory protein （KdpE） gene of Vibrio alginolyticus strain HY9901 ...The two-component regulatory system plays an important role in the growth and virulence of bacteria. In this study, the phosphate transcriptional regulatory protein （KdpE） gene of Vibrio alginolyticus strain HY9901 was cloned. Sequence analysis revealed that the length ofkdpE gene is 732 bp and encodes a putative protein of 243 amino acids. The predicted molecular weight （MW） of KdpE was 27.66 kD with an estimated pI of 5.01. Using SignalP 4.0 and TM- HMM Server 2.0 software, it was predicted that the KdpE protein was located in cytoplasm. It did not contain a signal peptide or a transmembranous region. A phylogenetic tree was constructed by MEGA 5.0 software, indicating that the KdpE of V. alginolyticus bad high genetic relationship with Vibrio campbellii and Vibrio parahaemolyticus. Using SWISS-MODEL work-space, the three-dimensional structures of conserved domain REC in the KdpE was determined. These results can provide a basis for further studies on the two-component regulatory systems of V. alginolyticus.展开更多
In this study, histidine kinase gene kdpD in the two-component regulatory system of Vibrio alginolyticus strain HY9901 was cloned for bioinformatics analysis. Sequence analysis revealed that kdpD gene （GenBank access...In this study, histidine kinase gene kdpD in the two-component regulatory system of Vibrio alginolyticus strain HY9901 was cloned for bioinformatics analysis. Sequence analysis revealed that kdpD gene （GenBank accession number： KJ544668） was 1 374 bp in length, encoding a putative protein of 457 amino acids. The predicted molecular weight （MW） of KdpE was 51.60 kD with a theoretical isoelectric point （pl） of 6.02. Using SignalP 4.0, TMHMM Server 2.0 and SoftBerry-Psite software, it was predicted that KdpE protein was equally located in Golgi apparatus, plasma membrane and endoplasmie reticulum （33.3%） , which did not contain a signal peptide but contained three transmembrane domains. KdpE protein had five casein kinase 1I phosphorylation sites, five protein kinase C phosphorylation sites and one cAMP- and cGMP-dependent protein kinase phosphorylation site. A phylogenetic tree was constructed by MEGA 5.0 software, which revealed that KdpD from V. alginolyticus had close genetic relationship with corresponding proteins from V. campbellii and V. parahaemolyticus. Using SWISS- MODEL Workspace, the three-dimensional structure of HATPase_c conserved domain in KdpD protein was constructed. These results may provide the basis for fur- ther studies on two-comoonent regulatory system KdpD/KdpE of V. alginolyticus.展开更多
[Objective]To clone araC gene of Vibrio alginolyticus HY9901 strain,and analyze bioinformatics.[Methods]the whole genome sequence of Vibrio alginolyticus on GenBank was used to design specific primers.According to the...[Objective]To clone araC gene of Vibrio alginolyticus HY9901 strain,and analyze bioinformatics.[Methods]the whole genome sequence of Vibrio alginolyticus on GenBank was used to design specific primers.According to the principle of PCR amplification sequence,the target gene araC was amplified,and then the sequence was further analyzed by bioinformatics method to establish the phylogenetic tree of araC gene and its corresponding subunit three-dimensional structure model.[Results]Sequence analysis revealed araC gene is 711 bp and encodes a putative protein of 236 amino acids.The predicted molecular mass of AraC was 26.92 ku.Using Signal P 4.0 and TMHMM Server 2.0 software for analysis,it was predicted that the AraC protein did not contain a signal peptide or a transmembranous region.The AraC protein had two cAMP and cGMP dependent protein kinase phosphorylation site,five protein kinase C phosphorylation sites,three casein kinase II phosphorylation sites,one prenyl group binding site(CAAX box)and five microbodies C-terminal targeting signal.The predicted results of protein subcellular localization showed that AraC was located in the mitochondria,nucleus and cytoplasm.Its protein is unstable and hydrophilic.The AraC protein is a transcriptional regulatory protein which belongs to HTH_18 superfamily.According to the prediction,secondary structure:a-helix(Alpha helix)accounted for 52.12%,random coil(31.78%),extended strand(11.02%),b-fold(Beta turn)accounted for 5.08%.V.alginolyticus,Vibrio parahaemolyticus and Vibrio palustris were clustered together,which implies that the genetic relationship between these three species was the closest.展开更多
[Objectives]To clone the sucC gene of Vibrio alginolyticus strain HY9901 and conduct the bioinformatics analysis.[Methods]Based on the sucC gene of V.alginolyticus strain HY9901,specific primers were designed to ampli...[Objectives]To clone the sucC gene of Vibrio alginolyticus strain HY9901 and conduct the bioinformatics analysis.[Methods]Based on the sucC gene of V.alginolyticus strain HY9901,specific primers were designed to amplify the full length sequence by PCR and make further analysis.[Results]The theoretical molecular weight of SucC protein was about 41528.45 Da,and the full length was 1167 bp,encoding 388 amino acids.It has no signal peptide and transmembrane region,and has a variety of functional sites.It is predicted that it is mainly located in the cytoplasm,and the ubiquitin and lactate modification sites overlap,and it has high gene homology with Vibrio parahaemolyticus.Theα-helix,random coil and extended strand are the main secondary structures.The similarity between the constructed three-level structure model and the template is high.[Conclusions]This study reveals the structural characteristics and functional potential of SucC protein,and provides a theoretical basis for the study of drug resistance mechanism and prevention strategies.展开更多
基金Natural Science Foundation of Guangdong Province(2025A1515011061)Outstanding Graduate Entering Laboratory Project of College of Fisheries,Guangdong Ocean University+1 种基金Undergraduate Innovation and Entrepreneurship Training Program of Guangdong Ocean University(CXXL2024007)Undergraduate Innovation Team of Guangdong Ocean University(CCTD201802).
文摘[Objectives] To analyze the function of cobQ gene from Vibrio alginolyticus strain HY9901,and to provide a reference for exploring the possible mechanism of cobQ gene from V.alginolyticus.[Methods] A pair of primers were designed based on the sequence of the V.alginolyticus cobQ gene and used to amplify the full-length gene by PCR.[Results] The PCR amplification results indicated that the cobQ gene has a full length of 780 bp,encoding 259 amino acid residues.The deduced amino acid sequence predicts a molecular weight of approximately 28.83 kD and an isoelectric point of 9.21.Sequence analysis revealed no N-terminal signal peptide cleavage site,suggesting the absence of both a signal peptide and transmembrane regions in this protein.The amino acid sequence contains 2 N-terminal myristoylation sites,1 N-glycosylation site,1 glycosaminoglycan attachment site,4 microbody C-terminal targeting signal sites,3 casein kinase II phosphorylation sites,and 4 protein kinase C phosphorylation sites.Subcellular localization prediction showed that the CobQ protein is primarily localized in the cytoplasm(65.2%probability).Homology analysis demonstrated that the amino acid sequence of the cobQ gene from V.alginolyticus shares up to 99%homology with other Vibrio species,clustering within the same subclade as Vibrio parahaemolyticus,indicating close phylogenetic relationships.Secondary structure prediction revealed proportions ofα-helices,random coils,and extended strands as 44.40%,36.68%,and 18.92%,respectively.The tertiary structure model exhibited 87.62%similarity to the template A0A165XBE1.1.[Conclusions] In this study,the V.alginolyticus cobq gene was successfully cloned and its sequence was analyzed by bioinformatics.It is expected to lay a foundation for the subsequent study of the regulatory mechanism of its protein on the virulence of V.alginolyticus.
基金Supported by Natural Science Foundation of Guangdong Province(2025A1515011061)Outstanding Graduate Entering Laboratory Project of College of Fisheries,Guangdong Ocean University+1 种基金Undergraduate Innovation and Entrepreneurship Training Program of Guangdong Ocean University(CXXL2024007)Undergraduate Innovation Team of Guangdong Ocean University(CCTD201802).
文摘[Objectives]This study was conducted to investigate the functional characteristics of the trxB gene in Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the trxB gene sequence of V.alginolyticus for PCR cloning of its full-length sequence.Systematic bioinformatics analyses were conducted to predict the physicochemical properties,secondary structure,and tertiary structure of the encoded protein.[Results]The trxB gene is 960 bp in length,encoding 319 amino acid residues.The deduced protein has a predicted molecular weight of 34.32 kDa and an isoelectric point(pI)of 4.77.Analysis of the amino acid sequence revealed a distinct signal peptide cleavage site at the N-terminus,with no transmembrane domains.The functional sites are as follows:1 N-glycosylation site,1 cAMP-and cGMP-dependent protein kinase phosphorylation site,4 protein kinase C phosphorylation sites,7 casein kinase II phosphorylation sites,1 tyrosine kinase phosphorylation site,11 N-myristoylation sites,1 prenyl group binding site,3 microbody C-terminal targeting signal sites,and 1 xanthine nucleotide-disulfide oxidoreductase class II active site.Subcellular localization prediction indicated the highest probability(44.4%)for endoplasmic reticulum localization.The TrxB amino acid sequence of V.alginolyticus shares 97.2%-98.4%homology with other Vibrio species,and they were clustered within the same subgroup.Secondary structure prediction showed proportions of random coils(31.97%),alpha-helices(31.66%),extended strands(25.08%),and beta turns(11.29%).The tertiary structure model exhibited 88.68%similarity to template 5vt3.1.A.[Conclusions]This study elucidated the characterization of the TrxB protein in V.alginolyticus,laying a theoretical foundation for the development of outer membrane protein subunit vaccines against this pathogen.
基金Supported by Natural Science Foundation of Guangdong Province(2025A1515011061)Outstanding Graduate Entering Laboratory Project of College of Fisheries,Guangdong Ocean University+1 种基金Undergraduate Innovation and Entrepreneurship Training Program of Guangdong Ocean University(CXXL2024007)Undergraduate Innovation Team of Guangdong Ocean University(CCTD201802).
文摘[Objectives]To develop a pair of specific primers for the PCR amplification of the full-length relA gene from Vibrio alginolyticus strain HY9901,as well as to conduct bioinformatics analysis.[Methods]The relA gene was amplified through PCR,and the resulting gene sequence was subsequently analyzed using bioinformatics tools,including amino acid sequence prediction,functional site analysis,subcellular localization prediction,and homology comparison.[Results]The relA gene had a total length of 2220 bp and encoded 739 amino acid residues.The molecular weight was approximately 84.1261 kDa,and its isoelectric point was 5.95.The protein lacked a signal peptide and transmembrane regions,while exhibiting multiple phosphorylation sites.Predictions regarding its subcellular localization suggested that it was predominantly situated in the cytoplasm.The amino acid sequence demonstrated a homology of 97%to 99%with other species within the genus Vibrio,and it clustered within the same subfamily as V.antiquarius and V.diabolicus.In the prediction of secondary structure,the proportions ofα-helix,extended strand,random coil,andβ-sheet were 54.13%,12.04%,28.15%and 5.68%,respectively.The similarity between the tertiary structure model and template 5kpw.1.w was 66%.[Conclusions]In this study,the relA gene of V.alginolyticus strain HY9901 has been successfully amplified and analyzed.The structural characteristics and potential functions of the encoded protein have been elucidated,thereby providing foundational data for understanding the role of this gene in V.alginolyticus.
文摘[Objective] The purpose of this study was to construct a vscC in-frame deletion mutant of Vibrio alginolyticus with no antibiotic resistance marker. [Method] The first vscC mutant molecules in vitro were generated by SOE-PCR and then lig- ated to a suicide vector pDM4 to construct a suicide recombinant vector pDM4- A vscC. To clone the recombinant vector, it was transformed to E. coli SY327 strain, and then positive clones were selected and proved by PCR analysis. After that, the pDM4-AvscC DNA was extracted in large numbers and transformed to the E. coil S17-1 strain that acted as a donor in bacterial conjugation using the heat shock method. The recombinant E. coli S17-1 strains then transferred the pDM4-AvscC to V. alginolyticus ZJ51-O by conjugation method; transconjugants were screened and selected sequentially using antibiotic selection strategy and sucrose based counter-selection system to find the suspected mutants wanted. Finally the putative mutants were identified by PCR and confirmed by sequencing analysis. [Result] ZJ51-OAvscC was successfully constructed. [Conclusion] This study laid foundation for further research on the function of vscC gene and molecular mechanism of type Ⅲ secretion system in V. alginolyticus. Simultaneously, by the effective method other unknown functional genes in V. alginolyticus genome would be researched.
基金supported by the National 863 Project（2012AA10A403）the Natural Science Foundation of Ningbo City of China（2014A610187）the Scientific Research Foundation of Graduate School of Ningbo University（G14041）
文摘Interleukin 1β（IL-1β）, the first interleukin to be characterized, plays a key role in regulating the immune response. In this study, we determined the c DNA and genomic DNA sequences of the IL-1β gene from the large yellow croaker, Larimichthys crocea. Phylogenetic analysis indicated that the IL-1β（Lc IL-1β） gene was most closely related to that of European seabass（Dicentrarchus labrax）, sharing 67.8% amino acid identity. In healthy large yellow croaker, Lc IL-1β transcription was detected in all tested tissues, with the highest level found in the head kidney. Upon Vibrio alginolyticus infection, Lc IL-1β transcription in all tested tissues was significantly upregulated. Intraperitoneal injection of recombinant Lc IL-1β（r Lc IL-1β） improved the survival rate and reduced the tissue bacterial load after V. alginolyticus infection. In addition, r Lc IL-1β induced monocytes/macrophages（MO/MΦ） chemotaxis and increased phagocytosis and bactericidal activity in vitro. These results suggest that Lc IL-1β plays an important role in the large yellow croaker immune response against V. alginolyticus.
基金Supported by the National Natural Science Foundation of China(No.41673076)the Major Agriculture Program of Ningbo(No.2017C110007)the K.C.Wong Magna Fund in Ningbo University
文摘A bstract Gut microbiota impacts the health of crustaceans. V ibrio alginolyticus is a main causative pathogen that induces the vibriosis in farmed swimming crabs, Portunus trituberculatus. However, it remains unknown whether gut bacteria perform functions during the progression of vibriosis. In this study, 16 SrRNA gene amplicon sequencing was used to investigate temporal alteration of gut bacterial community in swimming crabs in response to 72-h V. alginolyticus challenge. Our results show that V. alginolyticus infection resulted in dynamic changes of bacterial community composition in swimming crabs. Such changes were highlighted by the overwhelming overabundance of V ibrio and a significant fluctuation in the gut bacteria including the bacteria with high relative abundance and especially those with low relative abundance. These findings reveal that crab vibriosis gradually develops with the infection time of V. alginolyticus and tightly relates to the dysbiosis of gut bacterial community structure. This work contributes to our appreciation of the importance of the balance of gut bacterial community structure in maintaining the health of crustaceans.
基金This study was funded by the National Hi-Tech Development Plan("863"Plan)of China under contract Nos 2001AA5070 and 2002AA639600the Natural Science Foundation of Fujian Province under contract No.B0410022.
文摘Adhesion of Vibrio alginolyticus to the gill mucus of Pseudosciaena crocea has been investigated using [ methyl-^3 H ] thymidine as isotope tracer. The results showed that： the adhesive quantity of V. alginolyticus increased with bacterial concentrations and reached equilibrium after incubated for 180 min; the higher adhesive quantity was obtained at 15 ~ 30 ℃ and sourish conditions; adhesion of V. alginolyticus could not achieved without Na^＋ , and Ca^2＋ played an auxiliary role in the bacterial adhesion; adhesion of V. alginolyticus was inhibited remarkably by starvation, heat treatment and periodic acid treatment; all of the eight kinds of carbohydrates investigated enhanced the adhesion of V. alginolyticus to the gill mucus of P. crocea, among them, glucose, mannose, fructose and maltose showed the specially enhanced adhesion. The results indicated that E alginolyticus could adhere to the gill mucus of P. crocea facilely in seawater, and this bacterial adhesion was influenced by environmental factors and closely related to superficial carbohydrate structures and some heat-sensitive structures.
基金Supported by the National High Technology Research and Development Program of China (863 program, No. 2007AA09Z115)Technology Program of Xiamen (No. 3502Z73019)
文摘For the investigation of anti-infection immune response of Pseudosciaena crocea, 160 healthy fish samples were categorized into infected and control groups. Each individual fish in the infected group was injected intraperitoneally (i.p.) with 0.2 ml bacterial suspension of Vibrio alginolyticus in density of 2×107 CFU/ml, while each individual in the control group was injected i.p. with 0.2 ml sterile saline solution (0.85%). It was observed that the artificial injection of V. alginolyticus significantly increased the number of erythrocytes, leucocytes, lymphocytes in peripheral blood as well as peripheral serum antibacterial activity and antibody titer of large yellow croaker, and significantly reduced the number of peripheral blood granulocytes as compared with those in the control group. No significant difference in acid phosphytase and superoxide dismutase activity of serum was detected between the two groups. It is suggested that non-specific immune factors including leucocytes and anti-bacteria substance in peripheral blood played important role at the initial stage of infection, and specific immune factors such as antibody then played important role in response to anti-infection at the latter stage.
基金National Natural Science Foundation of China(32073015).
文摘[Objectives]The purpose was to investigate the function and mechanism of exsA gene in type III secretion system of Vibrio alginolyticus.[Methods]The full length of exsA was cloned using molecular biology techniques to analyze its biological information.Fluorescence quantitative PCR technology was used to analyze the expression of exsA after different media stress.[Results]The exsA gene contains an open reading frame(ORF)of 861 bp,encoding 286 amino acids.The physico-chemical analysis shows that the molecular structural formula is C1442H2267N393O441S12,the theoretical molecular weight is 32.549 kD,the theoretical pI value is 6.0,and the protein is non-hydrophilic and unstable.The gene does not contain a transmembrane region,and there is no obvious signal peptide.The prediction result of protein subcellular localization shows that the protein is inside the cell.The deduced amino acid sequence and constructed phylogenetic tree show that V.alginolyticus has a close relationship with Vibrio antiquarius.The qPCR results show that the expression level of exsA in different media is different,highest in TSB medium containing bile salts,followed by DMEM medium,and lowest in ordinary TSB medium.[Conclusions]The gene sequence,molecular structure and isoelectric point of exsA,as well as its expression in three different media were obtained.
基金Supported by Shenzhen Science and Technology Project(JCYJ20170818111629778,JCYJ20170306161613251)National Natural Science Foundation of Guangdong Province(2017A030313174)+2 种基金Natural Science Foundation of Guangdong Ocean University(C17379)Undergraduate Innovative and Entrepreneurial Team Project(CCTD201802)Science and Technology Program of Guangdong Province(2015A020209163)
文摘In this study, Va 1686 gene was cloned from Vibrio alginolyticus . The total length of the gene is 1 164 bp, and it could encode 387 amino acids. The physicochemical properties, protein structure, genetic evolutionary relationship and antigenic characteristics of the effect protein Va1686 of V. alginolyticus HY9901 type Ⅲ secretion system were studied and analyzed by bioinformatics methods and tools. The results showed that Va1686 is a stable hydrophilic and acidic protein without a transmembrane region and a signal peptide, and secondary structure to α-helix. The evolutionary analysis showed that V. alginolyticus HY9901 and V. harveyi were clustered together, which indicated that the genetic relationship between the two species was the closest. Va1686 contains a Fic superfamily conserved domain associated with cell division. Bioinformatics analysis showed that the B-cell preponderant epitopes of Va1686 might be localized in the regions of 48-49, 82-85, 125-126, 150-153, 185-186, 236-237 and so on. The 3D structure model of Va1686 subunit was simulated by SWISS-MODEL software and it was found that the vopS of V. parahaemolyticus was similar and the similarity was 89.46%. In this study, the feasibility of Va1686 as a common antigen of Vibrio was verified from the perspective of bioinformatics, which laid the foundation for the next step in vaccine development.
基金Supported by National Natural Science Foundation of China(3140234431572656)+1 种基金Project of Enhancing School with Innovation of Guangdong Ocean University(GDOU2015050216)Science and Technology Research Program of Guangdong Province(2014A020208117,2015A020209163)
文摘In this paper, the hopPmaJ gene, which encodes type III effector HopPmaJ of Vibrio alginolyticus, was cloned, sequenced and bioinformatically predicted. The results showed that hopPmaJ （GenBank accession number： KX245315） is 345 bp in length and encodes 114 amino acids, with a theoretical molecular mass of 12. 774 kD, and a pI of 4.45. The protein has multiple functional domains and binding sites, but no signal peptide. The tertiary structure of the protein is a homodimer. Based on multiple parameters, the possible dominant B cell antigenic epitopes of HopPmaJ were predicted to be at positions 13 - 16, 29 -30, 40 -42, 45 -49 and 84 -91.
基金Project for Outstanding Undergraduates Entering the Laboratory in Fisheries College of Guangdong Ocean UniversityNational Natural Science Foundation of China (32073015)+3 种基金Natural Science Foundation of Guangdong Province (2021A1515011078)Special Fund for Science and Technology Innovation Strategy of Guangdong Province(Scientific and Technological Innovation Cultivation of College Students)(pdjh2021b0239)Students’Platform for Innovation and Entrepreneurship Training Program of Guangdong Ocean University (CXXL2021122)Undergraduate Innovation Team Project of Guangdong Ocean University(CCTD201802)。
文摘[Objectives]To clone and analyze the TpiA gene of Vibrio alginolyticus HY9901.[Methods]According to the TpiA gene sequence of V.alginolyticus,a pair of specific primers was designed,and its full length was amplified by PCR.[Results]The full length of TpiA gene is 771 bp,encoding 256 amino acid residues in total,and the NCBI accession number is OM906798.According to the deduced amino acid sequence,its molecular weight was predicted to be about 26.97548 kDa,and its isoelectric point was 4.78.The amino acid sequence of the N-terminal signal peptide structure was predicted,and it was found that there was no obvious signal peptide cleavage site,no signal peptide,and no transmembrane region;the amino acid sequence contained 3 N-glycosylation sites,4 protein kinase C phosphorylation sites,2 casein kinase II phosphorylation sites,6 N-myristoylation sites,7 microbody C-terminal target signal site,and 1 triose phosphate isomerase active site.The prediction results of protein subcellular localization showed that TpiA may be located in mitochondria or cytoplasm,with probability of 39.1%and 34.8%,respectively.The amino acid sequence of the TpiA gene of V.alginolyticus shared 98.83%-99.61%homology with other Vibrio species,and it was clustered into the same subfamily with Vibrio parahaemolyticus and had a close relationship.In the secondary structure prediction,the proportions ofα-helix,random coil and extended chain were 44.53%,41.41%and 14.06%,respectively,and the similarity of its tertiary structure model to template 1aw1.1.A was 85.16%.[Conclusions]This study is intended to provide a basis for further research on the role of TpiA gene in the type III secretion system and related research on antibiotic resistance.
基金The Hi-Tech Research and Development Program ("863"Program) of China under contract Nos 2001AA635070 and 2002AA639600the Natural Science Foundation of Fujian Province of China under contract No.B0410022
文摘To get a better understanding of the starvation survival strategy of pathogenic Vibrio alginolyticus, log-phase cells were inoculated into sterile natural seawater for starvation studies. The results showed that all of total bacteria number, viable bacteria number and CFU number of V. alginolyticus increased remarkably at the initial starvation stage; after reaching their peaks at 5 d, both total bacteria number and viable bacteria number of V. alginolyticus fell slowly, while the CFU number fell more quickly after reaching its peak at 10 d; V. alginolyticus elongated their cells at the prophase of starvation, and then shrunk their volume and turned their shapes into ovals from rods at the anaphase of starvation; starved cells showed more sensitivity to heating and UV; starved cells showed no significant difference from unstarved ones at the lowest detection limit determined by indirect enzyme-linked immu- nosorbent assay （ELISA） ; starved cells＇ ability to adhere to the skin mucus of large yellow croaker （ Pseudosciaena crocea） showed a sharp decline as the starvation time increases; the cellular protein of V. alginolyticus increased remarkably at the ariaphase of starvation. The results indicated that pathogenic V. alginolyticus could survive in starvation for relatively long periods of time （ ≥2 months） in 28℃ natural seawater due to the morphological and physiological changes; however, starved V. alginolyticus cells showed less virulence and higher sensitivity under environmental stresses.
基金Supported by National Natural Science Foundation of China(No.31402344)Natural Science Foundation of Guangdong Province(S2013040014562)
文摘[ Objeetlve] This study aimed to investigate the role of dihydrolipoamide dehydrogenase （DLDH） in the pathopoiesis of Vibrio alginolyticua. [ Method] Using suicide plasmid pRE112 as the vector, d/dh deletion mutant strain ZJO3Adldh was constructed with insertion inactivation mutation method by Overlap PCR and homologous recombination technology to compare and analyze the differences in the growth, swimming ability, enzyme activity, biofilm formation and pathogenicity between wild-type strain ZJ03 and deletion mutant strain ZJO3△dldh. [ Result] dldh deletion mutant strain ZJ03 △d/dh exhibited prolonged lag phase, significantly reduced swimming ability （P 〈 0.05 ）, significantly reduced enzyme activity （P 〈 0.05 ）, significantly reduced biofilm formation ability （P 〈 0.05 ） and remarkably reduced pathogenicity to Epinephelus coioides （P 〈0.05） compared to wild-type strain ZJ03. [ Conclusion] This study provided theoretical basis for revealing the pathogenic mechanisms of Vibrio from the perspective of bacterial energy metabolism.
基金Supported by National Natural Science Foundation of China(31402344)Project of Enhancing School With Innovation of Guangdong Ocean University(GDOU2015050216)
文摘Vibrio alginolyticus, a gram-negative bacterium has been described to be one of the most common and economically important aquatic pathogens of fish and shellfish. Vaccine immunization is an effective approach preventing V. alginolyticus infection. Attenuated vaccine stimulates systemic immune response in the host, but few attenuated vaccine against V. alginolyticus is available. The type III secretion system （T3SS）, an important pathogenic factor of V. alginolyticus, is used by bacterial pathogens to inject effector proteins into the cytoplasm of their host cells. The T3SS forms a structure called needle complex with a multi-ring base that spans the bacteria and a needle-like extension that protrudes several nanometers from the bacterial surface, vscO locates at the ＂needle＂ site of T3SS, playing the role of escorting the molecular chaperone and effector proteins into host cells and further inducing the death of host cells. In this paper, an in-frame deletion mu- tant of vscO was constructed using overlap PCR and homologous recombination technology combining with chloramphenicol （Cm） and sucrose screening. The LDs0 changes of ZJO3AvscO mutant strains compared with the strain ZJ03 were e^amined in grouper （ Epinephelus coioides）. The ZJO3AvscO mutant showed about 150 times decrease in virulence in E. coioides compared with wide type ZJ03. After vaccination with ZJO3AvscO in E. coioides through injection and immersion, the spe- cific antibody titers were markedly higher than that in the saline control group （P 〈 0.05 ）. The titers of injection and immersion group on the forth week reached the maximums at 1：2 048 and 1： 128, respectively. The relative percentage survival （RPS） of injection group was 84%, while that in immersion group was 68%. These results indicate that the ZJO3AvscO of V. alginolyticus has a high immunogenicity, and can be used as live attenuated vaccine. In addition, RPS may be affected by vaccination and infection methods. This study can provide technical support for controlling fish diseases caused by V. alginolyticus.
基金Supported by Natural Science Foundation of Guangdong Province(2017A030313174)"Sail of the Sea-Starting Plan" of Guangdong Ocean University(qhjhzr201813)Innovation and Entrepreneurship Training Program for College Students(No.CXXL2019041)
文摘[Objectives]This study aimed to clone the tye A gene of Vibrio alginolyticus HY9901 strain and analyze its sequence by bioinformatics.[Methods] By referring to the entire genome sequence of Ⅴ.alginolyticus on Gen Bank,specific primers were designed.According to the principle of PCR amplification,the target gene tye A was amplified.By means of bioinformatics,the sequence of tye A was further analyzed,and the phylogenetic tree of tye A genes of Vibrio spp.and the corresponding subunit three-dimensional structure models were constructed.[Results] The length of the tye A gene of Ⅴ.alginolyticus strain HY9901 is 285 bp,and its theoretical molecular weight is 10.98 kD.According to prediction,there is no signal peptide or transmembrane region at the N-terminus of the sequence,and the amino acid sequence contains two casein kinase Ⅱ phosphorylation sites.The results of protein subcellular localization prediction show that the Tye A protein is located in the cell membrane.The protein is unstable and non-hydrophilic.The tertiary structure of Tye A protein of Ⅴ.alginolyticus is similar to that of Yersinia sp.According to prediction,Tye A has a major functional domain Pfam.In terms of secondary structure,alpha helix,random coil,extended strand and beta turn account for 85.11%,7.45%,4.26% and 3.19%,respectively.The homology of Tye A between Ⅴ.alginolyticus and Vibrio parahaemolyticus is up to 83%,so they are classified into one cluster.[Conclusions]This study will help to further understand the regulation mechanism of type Ⅲ secretion system in Ⅴ.alginolyticus.
基金National Natural Science Foundation of China(31402344,31572656)College Student Innovation Training Program(201510566042)+1 种基金Major Program for the Fundamental Research of the Department of Education of Guangdong Province(2014GKXM046)International Cooperation Innovation Platform Project of Universities in Guangdong Province(2013gjhz0008)
文摘The two-component regulatory system plays an important role in the growth and virulence of bacteria. In this study, the phosphate transcriptional regulatory protein （KdpE） gene of Vibrio alginolyticus strain HY9901 was cloned. Sequence analysis revealed that the length ofkdpE gene is 732 bp and encodes a putative protein of 243 amino acids. The predicted molecular weight （MW） of KdpE was 27.66 kD with an estimated pI of 5.01. Using SignalP 4.0 and TM- HMM Server 2.0 software, it was predicted that the KdpE protein was located in cytoplasm. It did not contain a signal peptide or a transmembranous region. A phylogenetic tree was constructed by MEGA 5.0 software, indicating that the KdpE of V. alginolyticus bad high genetic relationship with Vibrio campbellii and Vibrio parahaemolyticus. Using SWISS-MODEL work-space, the three-dimensional structures of conserved domain REC in the KdpE was determined. These results can provide a basis for further studies on the two-component regulatory systems of V. alginolyticus.
基金Supported by National Natural Science Foundation of China(31402344,31572656)"Sail in the Sea-Launch Plan"Science and Technology Innovation Project for College Students
文摘In this study, histidine kinase gene kdpD in the two-component regulatory system of Vibrio alginolyticus strain HY9901 was cloned for bioinformatics analysis. Sequence analysis revealed that kdpD gene （GenBank accession number： KJ544668） was 1 374 bp in length, encoding a putative protein of 457 amino acids. The predicted molecular weight （MW） of KdpE was 51.60 kD with a theoretical isoelectric point （pl） of 6.02. Using SignalP 4.0, TMHMM Server 2.0 and SoftBerry-Psite software, it was predicted that KdpE protein was equally located in Golgi apparatus, plasma membrane and endoplasmie reticulum （33.3%） , which did not contain a signal peptide but contained three transmembrane domains. KdpE protein had five casein kinase 1I phosphorylation sites, five protein kinase C phosphorylation sites and one cAMP- and cGMP-dependent protein kinase phosphorylation site. A phylogenetic tree was constructed by MEGA 5.0 software, which revealed that KdpD from V. alginolyticus had close genetic relationship with corresponding proteins from V. campbellii and V. parahaemolyticus. Using SWISS- MODEL Workspace, the three-dimensional structure of HATPase_c conserved domain in KdpD protein was constructed. These results may provide the basis for fur- ther studies on two-comoonent regulatory system KdpD/KdpE of V. alginolyticus.
基金National Natural Science Foundation of China(32073015).
文摘[Objective]To clone araC gene of Vibrio alginolyticus HY9901 strain,and analyze bioinformatics.[Methods]the whole genome sequence of Vibrio alginolyticus on GenBank was used to design specific primers.According to the principle of PCR amplification sequence,the target gene araC was amplified,and then the sequence was further analyzed by bioinformatics method to establish the phylogenetic tree of araC gene and its corresponding subunit three-dimensional structure model.[Results]Sequence analysis revealed araC gene is 711 bp and encodes a putative protein of 236 amino acids.The predicted molecular mass of AraC was 26.92 ku.Using Signal P 4.0 and TMHMM Server 2.0 software for analysis,it was predicted that the AraC protein did not contain a signal peptide or a transmembranous region.The AraC protein had two cAMP and cGMP dependent protein kinase phosphorylation site,five protein kinase C phosphorylation sites,three casein kinase II phosphorylation sites,one prenyl group binding site(CAAX box)and five microbodies C-terminal targeting signal.The predicted results of protein subcellular localization showed that AraC was located in the mitochondria,nucleus and cytoplasm.Its protein is unstable and hydrophilic.The AraC protein is a transcriptional regulatory protein which belongs to HTH_18 superfamily.According to the prediction,secondary structure:a-helix(Alpha helix)accounted for 52.12%,random coil(31.78%),extended strand(11.02%),b-fold(Beta turn)accounted for 5.08%.V.alginolyticus,Vibrio parahaemolyticus and Vibrio palustris were clustered together,which implies that the genetic relationship between these three species was the closest.
基金Supported by National Natural Science Foundation of China(32073015)Graduate Education Innovation Program of Guangdong Province(YJYH[2022]1)+1 种基金Undergraduate Innovation and Entrepreneurship Training Program of Guangdong Ocean University(CXXL2024007)Undergraduate Innovation Team of Guangdong Ocean University(CCTD201802).
文摘[Objectives]To clone the sucC gene of Vibrio alginolyticus strain HY9901 and conduct the bioinformatics analysis.[Methods]Based on the sucC gene of V.alginolyticus strain HY9901,specific primers were designed to amplify the full length sequence by PCR and make further analysis.[Results]The theoretical molecular weight of SucC protein was about 41528.45 Da,and the full length was 1167 bp,encoding 388 amino acids.It has no signal peptide and transmembrane region,and has a variety of functional sites.It is predicted that it is mainly located in the cytoplasm,and the ubiquitin and lactate modification sites overlap,and it has high gene homology with Vibrio parahaemolyticus.Theα-helix,random coil and extended strand are the main secondary structures.The similarity between the constructed three-level structure model and the template is high.[Conclusions]This study reveals the structural characteristics and functional potential of SucC protein,and provides a theoretical basis for the study of drug resistance mechanism and prevention strategies.
