河流弧菌(Vibrio fluvialis)对牙鲆(Paralichthys olivaceus)粘液的趋化和粘附作用

采用生长曲线法测定了河流弧菌在牙鲆表皮、鳃和肠粘液中的生长情况,结果表明,河流弧菌在3种粘液中都能生长,生长前期符合Logistic模型,然后进入Gompertz模型,在鳃粘液中的生长量高于肠粘液和表皮粘液。采用毛细管法研究了河流弧菌对牙鲆3种粘液的趋化作用,结果表明,河流弧菌对牙鲆3种粘液具有较强的趋化应答,在粘液总蛋白含量相同的情况下,河流弧菌对牙鲆鳃粘液的趋化应答显著强于对表皮粘液的趋化应答(P<0.05),而对肠粘液的趋化应答极显著低于对鳃粘液和表皮粘液的趋化应答(P<0.01)。采用3H-TdR示踪法进行了河流弧菌对牙鲆3种粘液的粘附作用研究,结果表明,河流弧菌对牙鲆表皮粘液和鳃粘液的粘附率较高,而对肠粘液的粘附率较低,特别是孵育30、60和90min时河流弧菌对牙鲆表皮粘液和鳃粘液的粘附率显著高于对肠粘液的粘附率(P<0.05)。这些结果表明,河流弧菌能够在牙鲆粘液,尤其是鳃粘液中生长和定植,有利于其进一步感染、致病。

河流弧菌-Ⅱ(Vibrio fluvialis-Ⅱ)噬菌体-VP_8的分离与研究

1996年夏季，从皱纹盘鲍脓疱病发病区采集海水样品46份，以皱纹盘鲍脓疱病病原菌河流弧菌-Ⅱ（Vibriofluvialis-Ⅱ）为指示菌，从这些样品中分离纯化到多株噬菌体，并对其中VP8进行了电子显微镜观察及生物学特性研究。其头部20面体的直径约57nm；尾鞘宽为18nm,长的33nm；尾轴宽约7nm，长约58nm；从形态上看属于BradlcyA型。

河流弧菌(Vibrio fluvialis)感染后牙鲆(Paralichthys olivaceus)血清抗菌物质的研究

采用1×107cells/ml的河流弧菌悬液背部肌肉注射感染牙鲆,注射后24h、48h试验组的血清抗菌活力分别极显著性(P<0.01)和显著性(P<0.05)高于对照组。用Sephadex G-25凝胶柱对第二次人工注射感染后24h牙鲆血清进行分离,其中第7—15收集管对河流弧菌具有抗菌活性,对大肠杆菌、枯草芽孢杆菌等指示菌也具有明显的抗菌作用,在4—100℃范围内,大部分活性分离组分随着温度的升高其抗菌活力有所增强。具有抗河流弧菌活性收集管(第9管)经弱阳离子交换柱分离后,OD280显示出2个蛋白吸收峰,具有抗河流弧菌活性的物质主要集中在第1吸收峰。SDS-PAGE分析显示经Sephadex G-25凝胶柱和弱阳离子交换柱分离的抗菌蛋白分子量较大。以上结果表明,牙鲆被河流弧菌感染后能很快产生大量抗菌物质释放到血清中;主要抗菌物质的分子量较大,且热稳定性较好;这些活性物质的抗菌活性具有较强的特异性。

海洋细菌Vibrio fluvialis的分离鉴定、产琼胶酶条件优化及酶的分离纯化

利用琼胶酶生产琼胶寡糖具有良好的应用前景,然而目前产琼胶酶菌株的性状不稳定、酶活力低等因素限制了琼胶酶的工业化应用。从龙须菜表面筛选出1株具有高琼胶酶活力的菌株A8,利用VITEK 2 GN微生物鉴定系统和16S rRNA基因序列分析,分别进行生理生化和分子生物学鉴定,确定该菌为Vibrio fluvialis A8。通过单因素试验和正交试验确定该菌的最适产酶培养基为:在人工海水中添加2 g/L琼脂、3 g/L半乳糖、3 g/L酵母浸粉、5 g/L Na Cl;最适培养条件为:接种量2%、温度20℃、p H 7.0。优化后发酵液琼胶酶的活力为21.80U/m L,是优化前的4.05倍。Pearson相关性分析表明,菌株生长对其产酶有正相关影响(P<0.01)。采用(NH4)2SO4分级沉淀和DEAE阴离子交换层析对琼胶酶进行分离纯化,琼胶酶的纯化倍数为3.26,比活力为141.52 U/mg,回收率为15.21%。SDS-PAGE凝胶电泳分析显示,琼胶酶的分子质量约为37 k Da。

Adhesion mechanisms of Vibrio fluvialis to skin mucus of Epinephelus awoara

Vibrio fluvialis incubated in trypticase soy broth(TSB)showed stronger adhesion to the skin mucus of Epinephelus awoara than V.fluvialis grown on trypticase soy agar(TSA),and this bacterial adhesion was assessed in terms of saturation kinetics.Treating bacteria with antibody against O-antigens resulted in significantly reduced bacterial adhesion.In the early growth stage,the adhering bacteria numbers increased with incubation time,peaked at 24 h,and then dropped sharply.Prior heat treatment of the mucus at various temperatures resulted in different effects on bacterial adhesion.No remarkable effect on bacterial adhesion was detected after treating mucus with protease K and trypsin,whereas periodic acid treatment significantly enhanced bacterial adhesion.Extracts of V.fluvialis surface structures at high concentrations significantly inhibited bacterial adhesion,while relatively low concentrations enhanced adhesion.V.fluvialis had strong affinity for a high molecular weight mucus component.These results indicate that V.fluvialis adhered strongly to E.awoara mucus by specific adhesion and that this adhesion was influenced by many factors,including bacterial conditions,mucus,and the environment.

Transcriptome analysis reveals immune-related genes in tissues of Vibrio anguillarum-infected turbot Scophthalmus maximus

Turbot Scophthalmus maximus is an important mariculture fish species with high economic value.However,the bacterial diseases caused by Vibrio anguillarum infection bring huge economic losses to the turbot aquaculture industry.To understand the immune response of the turbot against V.anguillarum infection and to explore novel immune-related genes,the transcriptome analysis of turbot spleen and gills were conducted after V.anguillarum infection.Differentially expressed genes(DEGs)were identified in spleen and gill of the turbot amounted to 17261 and 16436,respectively.A large number of immunerelated DEGs were enriched in cytokine-cytokine receptor interaction signaling pathway,and the others by the kyoto encyclopedia of genes and genomes(KEGG)enrichment.The gene ontology(GO)classification analysis revealed that V.anguillarum infection had the greatest effect on biological processes and cellular components.Twelve immune-related DEGs were identified in the spleen(cstl.1,egfl6,lamb21,v2rx4,calcr,and gpr78a)and gills(ghra,sh3gl2a,cst12,inhbaa,cxcl8,and il-1b)by heat map.The proteinprotein interaction(PPI)networks were constructed to analyze the immune mechanism.The results demonstrate that the maturation and antigen processing of major histocompatibility complex(MHC)class II molecule,and calcitonin-or adrenomedullin-regulated physiological activity were important events in the immunity of turbot against V.anguillarum infection.In the gills,the protein interactions in TGF-βsignaling pathway,production of inflammatory factors,and endocytosis regulation were most significant.Our research laid a foundation for discovering novel immune-related genes and enriching the knowledge of immune mechanisms of turbot against V.anguillarum infection.

基于核酸适配体的差减荧光法检测鳗弧菌(Vibrio anguillarum)

鳗弧菌(Vibrio anguillarum)可感染鳗鲡、虹鳟和大菱鲆等多种水产动物,是水产养殖中的一种重要病原菌,对其进行快速检测是病害防控的前提和基础。利用鳗弧菌与其核酸适配体之间有较强的亲和特异性,首次建立了一种基于核酸适配体的可定量检测鳗弧菌的差减荧光法。该方法对鳗弧菌有较好的特异性,对鳗弧菌的检测荧光值是其他菌(溶藻弧菌、哈维氏弧菌、铜绿假单胞菌、变形假单胞菌、嗜水气单胞菌、迟钝爱德华氏菌和大肠杆菌)的4~11倍,对鳗弧菌的最低检测限为102CFU/mL,可用于102~108CFU/mL的范围内的定量检测。通过对不同盐度海水和鱼体组织样品进行加标回收检测,结果表明,回收率和相对标准偏差等指标均符合相应的标准,说明该检测方法可用于海水样品和水产动物组织中鳗弧菌的检测。

Resveratrol Prevents Vibrio vulnificus-Induced Sepsis by Attenuating Necroptosis

Objective This study investigated how the natural phytophenol and potent SIRT1 activator resveratrol(RSV)regulate necroptosis during Vibrio vulnificus(V.vulnificus)-induced sepsis and the potential mechanism.Methods The effect of RSV on V.vulnificus cytolysin(VVC)-induced necroptosis was analyzed in vitro using CCK-8 and Western blot assays.Enzyme-linked immunosorbent assays and quantitative real-time polymerase chain reaction,western blot,and immunohistochemistry and survival analyses were performed to elucidate the effect and mechanism of RSV on necroptosis in a V.vulnificus-induced sepsis mouse model.Results RSV relieved necroptosis induced by VVC in RAW264.7 and MLE12 cells.RSV also inhibited the inflammatory response,had a protective effect on histopathological changes,and reduced the expression level of the necroptosis indicator pMLKL in peritoneal macrophages,lung,spleen,and liver tissues of V.vulnificus-induced septic mice in vivo.Pretreatment with RSV downregulated the mRNA of the necroptosis indicator and protein expression in peritoneal macrophages and tissues of V.vulnificusinduced septic mice.RSV also improved the survival of V.vulnificus-induced septic mice.Conclusion Our findings collectively demonstrate that RSV prevented V.vulnificus-induced sepsis by attenuating necroptosis,highlighting its potency in the clinical management of V.vulnificus-induced sepsis.

Protein Containing the GGDEF Domain Affects Motility and Biofilm Formation in Vibrio cholerae and is Negatively Regulated by Fur and HapR

Objective This study aimed to investigate whether the VCA0560 gene acts as an active diguanylate cyclase(DGC)in Vibrio cholerae and how its transcription is regulated by Fur and Hap R.Methods The roles of VCA0560 was investigated by utilizing various phenotypic assays,including colony morphological characterization,crystal violet staining,Cyclic di-GMP(c-di-GMP)quantification,and swimming motility assay.The regulation of the VCA0560 gene by Fur and Hap R was analyzed by luminescence assay,electrophoretic mobility shift assay,and DNase I footprinting.Results VCA0560 gene mutation did not affect biofilm formation,motility,and c-di-GMP synthesis in V.cholerae,and its overexpression remarkably enhanced biofilm formation and intracellular c-di-GMP level but reduced motility capacity.The transcription of the VCA0560 gene was directly repressed by Fur and the master quorum sensing regulator Hap R.Conclusion Overexpressed VCA0560 functions as an active DGC in V.cholerae,and its transcription is repressed by Fur and Hap R.

Immuno-Protective Efficiency of the Bivalent Inactivated Vaccine Against Vibrio scophthalmi and Aeromonas salmonicida Infections in Turbot(Scophthalmus maximus L.)

Vibrio scophthalmi and Aeromonas salmonicida can cause high turbot mortality and huge economic losses.Presently,vaccination is the most promising method for preventing communicable diseases.In this study,we used formalin to kill V.scophthalmi and A.salmonicida cells,and mixed with the mineralized oil adjuvant(Montanide^(TM)ISA 763 AVG)to prepare the bivalent inactivated vaccine.The results showed that turbot inoculated with the bivalent inactivated vaccine exhibited strong tolerance to the infection of V.scophthalmi and A.salmonicida,and no obvious clinical symptoms and pathological changes were observed.The activities of enzymes lysozyme,acid phosphatase and complement C3 had significantly increased after the vaccination.The antibody titer response of vaccinated turbot was greatly boosted,which was positively connected with the immunological impact according to ELISA results.Simultaneously,the expression levels of immune-related genes such as MHC-IIα,MHC-IIβ,CD4,CD8,TNF-αand IL^(-1)βwere up-regulated,demonstrating that it might stimulate humoral and cellular immunological response in turbot.These findings highlight the potential of the bivalent inactivated vaccine for controlling V.scophthalmi and A.salmonicida infections in turbot.

Monitoring and analysis of contamination of Vibrio parahaemolyticus and Vibrio alginolyticus in seafood in Haikou

Objective:To investigate the contamination and distribution of Vibrio parahaemolyticus and Vibrio alginolyticus in seafood in Haikou City.Methods:Three types of seafood sold in Haikou from 2020 to 2022 were collected,Vibrio parahaemolyticus and Vibrio alginolyticus were detected according to the National Food Safety Standard Food Microbiological Examination of Vibrio parahaemolyticus(GB 4789.7-2013),and they were identified by real-time fluorescence PCR.The detection of Vibrio parahaemolyticus and Vibrio alginolyticus in different kinds of seafood,different years and different quarters was analyzed.Results:A total of 119 seafood samples were collected.Among them,24 samples were positive with Vibrio parahaemolyticus,with a positive rate of 20.1%;46 samples were positive with Vibrio alginolyticus,with a positive rate of 38.7%.Among various types of seafood,shrimp have the highest positivity rate for Vibrio parahaemolyticus at 50%,while shellfish have the highest positivity rate for Vibrio alginolyticus at 48%.Comparing between monitoring years,the positive rate of Vibrio alginolyticus was the highest in 2021(76.7%),while the positive rate of Vibrio parahaemolyticus was the highest in 2022(25%).Comparing between different quarters,the positivity rate for Vibrio alginolyticus was found to be highest in the second quarter at 80%,while the positivity rate for Vibrio parahaemolyticus was highest in the fourth quarter at 33.3%.There were statistically significant differences(P<0.05)in the positivity rate for Vibrio alginolyticus in different years and quarters,as well as in the positivity rate for Vibrio parahaemolyticus in different types and quarters.Conclusion:Vibrio parahaemolyticus and Vibrio alginolyticus were found in seafood products in Haikou City from 2020 to 2022.It is recommended that relevant departments strengthen supervision to ensure the safety of seafood products consumed by the public.

Characterization of Caspase Gene Family Members in Spotted Sea Bass(Lateolabrax maculatus)and Their Expression Profiles in Response to Vibrio harveyi Infection

The caspase gene family is a crucial gene cluster that regulates apoptosis which contribute to programmed cell death,cell proliferation and differentiation,and several immune responses.In our study,a complete set of 12 caspase genes were identified in spotted sea bass Lateolabrax maculatus.These genes were divided into three subfamilies:2 inflammatory caspases(casp-1 and casp-14-like),5 apoptosis initiators(casp-2,casp-8a,casp-8b,casp-9,and casp-10),and 5 apoptosis executioners(casp-3a,casp-3b,casp-3-like,casp-6,and casp-7).Their phylogenetic relationships,synteny and gene structures were systematically analyzed.Furthermore,the relative expression profiles of the caspase family members in the liver,intestine,head kidney,and spleen were measured by q PCR after infection with Vibrio harveyi.The results showed that the overall mRNA levels of the caspase genes were dramatically increased after V.harveyi infection,and the expression patterns varied among genes and tissues.More caspase genes underwent pronounced expression changes in the head kidney and spleen than in the liver or intestine,mainly after 48 h of the challenge.Specifically,casp-3a,casp-3b,casp-3-like,casp-6,casp-7,casp-8a,casp-8b,casp-10,and casp-14-like in the head kidney,and casp-3-like,casp-6,casp-7,and casp-14-like in the spleen,were the most responsive caspase genes which may contribute significantly to immune regulation in spotted sea bass.Additionally,the apoptosis level in head kidney and spleen after infection were examined using the Caspase assay.Our study provides a systemic overview of the caspase gene family in spotted sea bass after V.harveyi infection and lays a foundation for further deciphering the biological roles of these caspase genes.

Identification of suitable reference genes for quantitative gene expression analysis in clam Cyclina sinensis under salinity stress and Vibrio infection

The appropriate reference gene is a prerequisite for accurate normalization of gene expression level,and research on suitable reference genes in clam Cyclina sinensis is scarce.To improve the situation,we selected five commonly used housekeeping genes,including β-actin,Elongation factor 1-α(EF1-α),Glyceraldehyde-3-pho sphate dehydrogenase(GAPDH),40S ribosomal protein S18(RPS18),and Tubulin a(TUB-α),then evaluated their expression stability in different adult tissues and under different experimental treatments(salinity stress and Vibrio parahaemolyticus infection).Their expression stability was analyzed by three frequently used programs,geNorm,NormFinder,and BestKeeper.This analysis indicated that multiple genes should be used for normalization,and we concluded that the reference gene combination GAPDH-RPS18-β-actin,should be used for qRT-PCR analysis in different tissues of C.sinensis under normal physiological conditions.For the clams under salinity stress and Vibrio infection,EF1-α-GAPDHRPS18 was recommended as the gene combination for qRT-PCR normalization.TUB-αwas generally poorly ranked by all programs,and should not be used in future studies.This study should provide fundamental support for accurate quantitative gene expression analysis of this species.

Virulence changes in Vibrio parahaemolyticus during the freezing of Penaeus chinensis

Although Vibrio parahaemolyticus has become the most common pathogen in fresh and frozen seafood,its virulence changes have often been ignored during the processing of seafood.To investigate these potential risks,we used frozen Penaeus chinensis as examples,and the most virulent factors of V.parahaemolyticus,including amounts,viable but nonculturable(VBNC)status,toxins TDH and TRH,and virulence genes tdh and trh,were determined.Bacterial quantities were signifi cantly reduced during drain and sprinkling phases,but caused by different factors.By SYTO9 and PI staining showed that washing was the main reason for the bacterial reduction at the drain phase,while the strain entering VBNC state was another reason at sprinkling phase.Their hemolysis toxicity,produced by TDH and TRH,became stronger after inoculation on shrimp,and could be detected throughout the process.Moreover,tdh and trh also exhibited trends similar to that of the hemolysis toxicity test.tdh was almost to a two-fold expression level during ice-glazing phase,while trh only express at a low level,less than half of the expression level before inoculation.These results demonstrated that the strains were not dead during freezing process,but became VBNC cells,which still produced and accumulated toxins,especially TDH,the most virulent factor.

Proteomic and Functional Analyses of Outer Membrane Vesicles Secreted by Vibrio splendidus

Vibrio splendidus is an important opportunistic pathogen ubiquitously present in the marine environment,exhibiting virulence to a variety of cultured animals.The extracellular products secreted by V.splendidus are crucial to bacterial survival and virulence.In this study,the secretion of outer membrane vesicles(OMVs)by V.splendidus was determined,purified,and morphologically characterized.The protein composition of OMVs was analyzed by proteomic analysis.The results showed that approximately 120 proteins were contained in these OMVs,including outer membrane proteins,flagellins,ABC transporters,protease,and iron regulation proteins,etc.,which were involved in bacterial motility,formation of biofilms and the cell membrane components,and cellular localization based on their structural molecule activity,passive transmembrane transporter activity,channel activity,neurotransmitter receptor activity,extracellular ligand-gated ion channel activity,glutamate receptor activity,ligand-gated ion channel activity,and transmembrane signaling receptor activity.To explore the biological functions of OMVs in V.splendidus,the effects of OMVs on the bacterial adaption to iron limitation,antibiotic,and the coelomic fluid of the Apostichopus japonicus were confirmed.This study is the first time to show that V.splendidus secretes OMVs,and OMVs carry functional proteins that enhance bacterial survival under various stresses.

Amplification and Bioinformatics Analysis of h-ns Gene of Vibrio alginolyticus

[Objectives]To amplify the h-ns gene of Vibrio alginolyticus and analyze it by bioinformatics.[Methods]According to the h-ns gene sequence of V.alginolyticus HY9901,a pair of specific primers were designed and amplified by PCR.[Results]The h-ns gene was 408 bp in length and 135 amino acids were encoded.The predicted theoretical protein molecular weight was about 14.98 kD,and the isoelectric point was 4.99.Protein subcellular localization,SignalP 5.0,TMHMM Server 2.0 and SoftBerry-Psite predictions showed that H-NS was located outside the cell membrane,and the protein was unstable and hydrophobic.There was no signal peptide cleavage site,no transmembrane region and no KEGG metabolic pathway.The amino acid sequence contained three phosphorylation sites,one N-terminal myristoylation site and three microsomal C-terminal target signal sites.Using MEGA 5.0,H-NS phylogenetic tree was constructed by ortho-connection method.The results showed that H-NS of V.alginolyticus was closer to H-NS of Vibrio diabolicus.Using SWISS-MODEL,the three-dimensional structure model of H-NS subunit was simulated,which was similar to the crystal structure of Salmonella typhimurium H-NS1-83.[Conclusions]This study lays a foundation for exploring the regulation mechanism of V.alginolyticus H-NS protein on bacterial virulence in the future.

Effects of Microplastics on Expression of Resistance Genes and Virulence Genes of Vibrio alginolyticus

[Objectives]To study the effects of microplastics on antibiotic resistance genes and virulence genes of Vibrio alginolyticus,so as to provide a certain reference for controlling marine pollution,curbing the spread of environmental antibiotic resistance genes and virulence genes,formulating environmental policies,and maintaining food safety.[Methods]After adding V.alginolyticus into the artificial seawater,they were divided into three groups,namely blank control group(BLK),polyvinyl chloride microplastic group(PVC group)and polyvinyl alcohol microplastic group(PVA group).Aerated culture experiments were carried out,and the effects of microplastics on the expression of resistance genes and virulence genes of V.alginolyticus were studied by PCR and qPCR methods.[Results]The presence of microplastics significantly changed the resistance gene structure of V.alginolyticus.Compared with the control group,the cfxA and cfr resistance genes were detected in the microplastic group.However,only PVC group detected blaZ resistance gene,and only PVA group did not detect aaC resistance gene.In addition,compared with the control group,the expressions of virulence genes in the microplastic group were all down-regulated(P<0.01).[Conclusions]This study provides some reference for curbing the spread of environmental antibiotic resistance genes and virulence genes,formulating environmental policies,and maintaining food safety,but the specific mechanisms of drug resistance and virulence need further research.

Cloing and Bioinformatics Analysis of ndk Gene from Vibrio alginolyticus

[Objectives]The paper was to clone and analyze bioinformatics of ndk gene from Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the ndk gene sequence of V.alginolyticus HY9901.The full length of ndk gene was amplified by PCR and bioinformatics analysis was performed.MEGA 5.0 software was used to construct NDK phylogenetic tree by neighbor-joining method.SWISS-MODEL program was used to obtain the three-dimensional structural model of single subunit from NDK protein.[Results]The ndk gene,molecular structural formula C702H1094N192O214S7,was 426 bp in total,encoding 141 amino acids,with the theoretical molecular weight of 15.87199 kD and the theoretical pI value of 5.13.The prediction results of protein subcellular localization,SignalP 5.0,TMHMM Server 2.0 and SoftBerry-Psite showed that NDK mainly existed in the cytoplasm,and the protein was unstable and hydrophobic.There was neither signal peptide cleavage site,nor transmembrane region and KEGG metabolic pathway.The amino acid sequence had two protein kinase C phosphorylation sites,a casein kinaseⅡphosphorylation site,a N-myristoylation site,three microbody C-terminal target signal sites,and a nucleoside diphosphate kinase active site.Homology analysis showed that the NDK of V.alginolyticus had high homology with that of V.diabolicus,with a similarity of 98.58%.Analysis of the structural functional domain revealed that the protein had one NDK structural functional domain.The prediction results of secondary structure showed that theα-helix,random coil,β-sheet and extended strand accounted for 53.19%,28.37%,7.09%and 11.35%,respectively.Analysis of NDK protein via STRING database demonstrated that the proteins interacting with NDK protein were NrdA,NrdB,GmK,CmK,TmK,PyrG,PyrH,RelA,FolE and SpoT.[Conclusions]The study plays a positive role in the prevention and control of vibriosis and the improvement of the current aquaculture environment.

Cloning and Bioinformatics Analysis of vscB Gene of T3SS Chaperone of Vibrio alginolyticus

[Objectives]To clone and analyze the vscB gene of Vibrio alginolyticus HY9901 by bioinformatics.[Methods]A pair of specific primers were designed according to the vscB gene sequence of Vibrio alginolyticus HY9901.The full length of the primers was cloned by PCR and analyzed by bioinformatics.[Results]The vscB gene was 429 bp long,encoding 142 amino acids,with a theoretical molecular weight of 16.4 kDa and a pI value of 5.48.Amino acid sequence analysis of VscB showed that VscB was not a secretory protein,without signal peptide and transmembrane region,and there were protein kinase C phosphorylation site and casein kinase II phosphorylation site in the sequence.Homologous comparison of amino acid sequences showed that VscB of V.alginolyticus had the highest protein similarity with Vibrio Parahaemolyticus,reaching 91%.Phylogenetic tree analysis showed that the corresponding proteins of V.alginolyticus VscB,Vibrio Parahaemolyticus and Vibrio diabolicus were clustered in the same subfamily.Functional domain analysis showed that it had CesT family domain.Tertiary structure prediction showed that there were 3α-helices and 5β-turns in VscB protein.[Conclusions]This study provided a theoretical basis for further study on the function of chaperone of V.alginolyticus.

Amplification and Bioinformatics Analysis of vscN Gene from Vibrio alginolyticus

[Objectives]The paper was to clone and analyze bioinformatics of vscN gene from Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the vscN gene sequence of V.alginolyticus HY9901.The full length of vscN gene was amplified by PCR and bioinformatics analysis was performed.[Results]The vscN gene was 1323 bp in total,encoding 440 amino acids,with the theoretical molecular weight of 47.86 kD and the theoretical pI value of 5.89.The online prediction showed that there was no signal peptide and no transmembrane region in VscN.The amino acid sequence had 10 N-myristoylation sites,8 phosphorylation sites(2 protein kinase C phosphorylation sites,6 casein kinase II phosphorylation sites),1 amidation site,11 microbody C-terminal target signal sites,1 ATP/GTP binding site motif A(P ring),and 1 ATPaseαandβsubunit specific site.Homology analysis showed that the VscN protein of V.alginolyticus had high homology with that of V.antiquarius,with a similarity of 95.14%.Phylogenetic tree analysis showed that the VscN of V.alginolyticus was clustered into the same subgroup as that of V.diabolicus and V.antiquarius.Functional domain analysis of VscN protein showed that it had Pfam and AAA domains,and involved in the regulation of bacterial virulence.The three-dimensional structure model of VscN simulated by SWISS-MODEL software was similar to the structure of flagellate-specific ATPase FliH-FliI complex.[Conclusions]The results lay a foundation for further study on the regulatory mechanism of VscN protein on bacterial virulence.