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.

病原哈维氏弧菌(Vibrio harveyi)TS-628菌株基因组分析

哈维氏弧菌是海洋鱼类和无脊椎动物的重要条件致病菌,经常造成海水养殖动物严重病害,给水产养殖业造成巨大的经济损失,而不同来源的哈维氏弧菌的毒力存在很大差异。本研究采用第二代Illumina Hiseq平台与第三代PacBio平台相结合的测序技术,对分离自福建海域患病青石斑鱼的哈维氏弧菌TS-628菌株进行基因组测序和分析,挖掘该菌的基因组信息,从基因组层面了解该病原菌的毒力相关基因,以期为哈维氏弧菌后续致病机制的研究以及哈维氏弧菌病防治提供有力数据支撑。结果表明,哈维氏弧菌TS-628菌株基因组含2条染色体、2个质粒,其中1个质粒为新发现质粒;基因组大小为6151198 bp,基因组GC含量为44.68%,共编码5658个基因,共预测到毒力基因747个,毒力基因主要与细菌的黏附、摄铁系统及分泌系统有关;此外,还发现耐药基因294个,反映该菌株不仅具有编码多种毒力因子的潜力,同时具有抗多种药物的可能。

两株珍珠龙趸病原性哈维弧菌(Vibrio harveyi)的分离与鉴定

自海南陵水和广东深圳养殖场患病珍珠龙趸(pearl gentian)分离到2株优势菌(X12XC30和X13SZ03),经回归感染实验证实2株菌是珍珠龙趸的病原菌,对珍珠龙趸的半致死浓度(LD50)分别是6.58×106 CFU·g-1和9.83×105 CFU·g-1。对菌株进行形态、颜色和生长条件等常规生理生化实验,并进行16S r DNA,rct B和tox R等管家基因的序列分析综合鉴定。结果显示:2株病原菌均为短杆状革兰氏阴性菌,TCBS生长为黄色,对O/129(150 ug·片-1)敏感,生理生化指标与哈维弧菌标准株一致;16S r DNA,rct B和tox R序列在Genbank上检索与哈维弧菌的同源性最高,根据管家基因的序列构建系统进化树,自然地与哈维弧菌分支聚类为一支,确定这2株菌均为哈维弧菌。药敏实验发现2株哈维弧菌均耐呋喃唑酮,而对诺氟沙星、恩诺沙星、环丙沙星和氯霉素等药物敏感。哈维弧菌具有较高的致死率,在珍珠龙趸养殖业中存在潜在的威胁。

In vitro Inhibitory Activity of Shisandra chinensis and Polygonatum sibiricum against Vibrio harveyi and Its Biofilms

[Objective] This study aimed to analyze the in vitro inhibitory activity of Shisandra chinensis and Polygonatum sibiricum against Vibrio harveyi and its biofilms. [Result] By agar diffusion test, in vitro inhibitory activity of 5. chinensis and P. sibiricum against V. harveyi was investigated. The minimal inhibitory concentration （ MIC） and minimal bactericidal concentration （MBC） of 5. chinensis and P. sibiricum against V. harveyi were determined by doubling dilution meth-od. The inhibitory activity of 5. chinensis and P. sibiricum on the formation of V. harveyi biofilms was evaluated by modified MTT assay. [ Result ] Both 5. chinen-sis and P. sibiricum had inhibitory activity against V. harveyi. The inhibition zone diameter of 5. chinensis against V. harveyi was 17. 95 mm; MIC and MBC of 5. chinensis were both 3.125 mg/ml. The inhibition zone diameter of P. sibiricum against V. harveyi was 12. 22 mm; MIC and MBC of P. sibiricum were 3.125 and 6.250 mg/ml, respectively. When the concentration was higher than 6. 25 mg/ml, 5. chinensis decoction had extremely significant inhibitory activity against V. harveyi （P 〈 0. 01） ; when the concentration was higher than 3. 125 mg/ml, P. sibiricum had extremely significant inhibitory activity against V. harveyi （P 〈0. 01）. [ Conclusion] 5. chinensis and P. sibiricum could significantly inhibit V. harveyi and its biofilms.

Disruption of chemotaxis-related genes affects multiple cellular processes and the virulence of pathogenic Vibrio harveyi

Chemotactic motility is involved in the virulence of many pathogenic bacteria. In order to understand the role of chemotactic motility of Vibrio harveyi in cellular processes and virulence, mini-TnlO/Kan transposon-induced mutants with deficient chemotactic motility were constructed, screened, and iden- tified. Sequence analysis revealed that the 465-bp fragment （GenBank accession number HM630274） fank- ing the transposon insertion site in mutant TS-CM1 had the highest identity （96.9%） with a hypothetical protein gene of V. harveyiATCC BAA-1116 and the second-highest identity （91.8%） with the pgk gene of V. parahaemolyticus RIMD 2210633. In another mutant, TS-CM2, 356 bp of transposon-flanking sequence （GenBank accession number HM630275） also showed the highest identity （94.6%） with a hypothetical pro- tein gene of V. harveyi ATCC BAA-1116 and the second-highest identity （92.4%） with the flaB gene of V. alginolyticus HY9901. Studies on virulence-related biological characteristics such as growth, motility, adhe- sion, and infectivity of the mutants showed that disruption of either the flagellin gene or energy metabolism gene led to subsequent loss of chemotactic motility and changes in growth, motility, adhesion, and viru- lence of the pathogenic E harueyi. Hence, the flagellin gene and crucial energy metabolism gene played an important role in the chemotactic motility of V. harveyi.

Immunogenicity and protective efficacy of Vibrio harveyi pcFlaA DNA vaccine in Epinephelus awoara

The FlaA gene from Vibrio harveyi marker, was cloned into the eukaryotic expression with a short nucleotide sequence encoding the Flag vector pcDNA3.1（＋） （designated as pcFlaA）. Ninety grouper （Epinephelus awoara） were separated into three equal size groups. An experimental group was immunized with pcFlaA, Control I group was immunized with the vector pcDNA3.1（＋）, and Control 1I group was immunized with PBS. The expression of pcFlaA mRNA and protein was examined using reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemistry. We also evaluated the immunogenicity and protective efficacy of pcFlaA against V. harveyi by measuring the lymphocyte proliferation response and serum levels of specific antibody and conducting a bacterial challenge test. We successfully transfected the fish muscle with pcFlaA. The pcFlaA mRNA and protein was expressed in the muscle cells for up to one month following injection. The proliferation response of lymphocytes in fish immunized with pcFlaA was significantly higher than in control group II. Furthermore, the immunized fish generated specific antibody. The vaccination also resulted in significantly higher survival during the bacterial challenge test.

Transcriptional responses to starvation of pathogenic Vibrio harveyi strain DY1

Vibrio harveyi is a pathogen of various aquatic organisms that has been recently associated with massive mortality episodes in the aquaculture industry.Recurrent outbreaks of vibriosis are closely correlated with the capacity of this bacterial species to survive long-term starvation conditions.To study the regulation mechanism of gene expression at the transcriptional level in V.harveyi under starvation conditions,the transcriptomic response profiles were determined of the Portunus trituberculatus pathogen V.harveyi strain DY1 under normal conditions and after four weeks of starvation.A total of 4679 and 4661 genes were expressed in the non-starved and starved cells,respectively.The significantly differentially expressed genes(DEGs)between non-starved and starved groups were identified,in which 255 genes were up-regulated and 411 genes were down-regulated.GO analysis and KEGG enrichment analysis were used to analyze the DEGs and revealed the involvement of these DEGs in many pathways,including ABC transporters,flagellum assembly,and fatty acid metabolism.Several DEGs were randomly selected and their expression levels were confirmed by quantitative real-time PCR(qRT-PCR).This is the first comprehensive transcriptomic analysis of starvation ef fects in V.harveyi.Our findings will facilitate future study on stress adaptation and survival mechanisms of V.harveyi.

An Extracellular Oligopeptide Permease May Be a Potential Virulence Factor of Vibrio harveyi

An oligopeptide permease A(OppA)was purified from the extracellular product of Vibrio harveyi SF-1.The molecular weight of the purified protein was estimated to be 58 kDa on SDS-PAGE.The purified protein showed phospholipase C activity at the optimal values of temperature 50℃ and pH 8.0.The enzymatic activity decreased when the temperature increased to 40℃.The N-terminal sequence of the purified protein was determined as ADVPAGTKLA,which is similar to that of OppA.The OppA pre-cursor gene was cloned from the genome of V.harveyi SF-1.The gene consisted of 1665 base pairs and encoded a 554 amino acid polypeptide,which showed a high similarity to those of the OppAs of V.harveyi and other Vibrio species.The gene was subcloned into pET-28a(+)and expressed in Escherichia coli.The expressed recombinant protein was purified by Ni-NTA metal affinity chro-matography.The expressed recombinant protein showed a 58 kDa band on SDS-PAGE and exhibited phospholipase C activity with the optima of temperature 50℃ and pH 8.0.The purified protein was toxic to the flounder gill cells.An OppA mutant of V.harveyi SF-1 was constructed by homologous recombination.The mutant strain was less virulent when it was intraperitoneally inoculated to zebra fish,with the LD50 of 5.46×105 CFU fish-1,compared to 3.11×104 CFU fish-1 of the wild-type strain,which indicated that the OppA might play an important role in the pathogenicity of V.harveyi.

Antimicrobial peptide hepcidin contributes to host defense of Centropristis striata against Vibrio harveyi challenge

Hepcidins are small cysteine-rich antimicrobial peptides that play a vital role in immunity against pathogen invasion.Here,a hepcidin(Cshep)from Centropristis striata was described,which is considered as a valuable aquaculture marine species in China.The open reading frame consisted of 273 bp.Eight conserved cysteine residues were identified.Phylogenetic analysis showed that Cshep had a relatively close relationship with the hepcidin from Epinephelus moara.Quantitative real-time PCR analysis demonstrated that Cshep was highly expressed in liver and significantly up-regulated when challenged with Vibrio harveyi.In addition,the synthetic Cshep peptide had a high antimicrobial activity against V.harveyi,but low against other pathogenic bacteria tested in this study.The killing kinetics analysis revealed that Cshep had a fast bactericidal effect on V.harveyi.These results suggested that Cshep may be involved in the immune response of C.striata against V.harveyi infection.

非可培养状态哈维氏弧菌(Vibrio harveyi)复苏后生理特征及毒力相关基因表达

将哈维氏弧菌SF1接种在自然海水中低温诱导进入活的非可培养状态,用添加营养物质后升温培养的方法进行复苏。用透射电镜观察不同状态哈维氏弧菌细胞形态和结构,研究哈维氏弧菌复苏后对不良环境的抵抗力,用RT-PCR检测丝氨酸蛋白酶基因Ser和rpoS基因的表达。结果表明,进入VBNC状态的哈维氏弧菌由杆状变为球状,体积比正常细胞小,复苏后恢复正常形态且对紫外辐射,热激的抵抗能力没有明显改变,对高渗透压的抵抗力减弱,而对冷激的抵抗力增强。复苏后的哈维氏弧菌对抗生素的敏感性没有改变。用RT-PCR未检测到在VBNC状态时Ser和rpoS基因的表达,而在复苏后的细胞均检测到该基因的表达,可能是其保持致病性的重要原因之一。

Bioluminescence and Chitinase Production during Chitin Fermentation by Vibrio harveyi

Vibrio harveyi, like other luminescent bacteria, is capable of producing extracellular chitinases. Microbial chitinases are utilized to depolymerize chitin into chitooligosaccharides and N-acetylglucosamine for the acquisition of carbon and possibly nitrogen, needed for survival. For many luminous marine bacteria （Vibrio spp.）, quorum-sensing is highly speculated to be responsible for bioluminescence; however, in terrestrial species （Photorhabdus spp.） luminosity seems to be controlled through unknown mechanism of phase variation. In the present work, the correlation between bacterial luminosity and chitinase production of F. harveyi was studied. The utilization of bioluminescence could prove to be an easier and more convenient method to monitor chitin fermentations that employ luminous bacteria. Results from the fermentation study indicate that luminosity of F. harveyi inversely correlates with chitinase production. In other words, during chitin fermentation, chitinase production was seen to increase while luminosity decreased with respect to growth and growth conditions. Furthermore, the results also suggest that V. harveyi may utilize an alternate mechanism that can counter quorum-sensing mechanisms to ensure bacterial survival under deteriorating growth conditions. The inverse relationship observed in this study may lead to a basic understanding of monitoring and studying chitin fermentations and anti-quorum-sensing/phase variation mechanisms exhibited by luminous bacteria.

哈维氏弧菌(Vibrio harveyi)ML01株胞外产物及分泌性蛋白的分离与特性分析

本研究以引起珍珠龙胆石斑鱼[Epinephelus fuscoguttatus(♀)?Epinephelus lanceolatus(♂)]幼鱼"皮肤溃疡病"的哈维氏弧菌(Vibrio harveyi)ML01株为研究对象,采用平板膜覆盖技术和柱层析技术,分离、纯化了ML01株的胞外产物及分泌性蛋白。应用毒性试验、质谱分析与分子克隆技术,对纯化的胞外产物和3种主要的分泌性蛋白进行了特性分析与鉴定。结果显示,哈维氏弧菌ML01株的胞外产物(Extracellular products,ECPs)具有酯酶、明胶酶、淀粉酶、酪蛋白酶活性,无脲酶活性。ECPs对羊红细胞无溶血性,对斑马鱼(Danio rerio)的半数致死剂量(LD50)为19.55μg/g鱼体重。从ML01株中分离到3种主要的分泌蛋白P42、P36、P31,其分子量分别为42、36、31 k Da。经质谱鉴定和分析,这3种蛋白分别为哈维氏弧菌的外膜蛋白Omp U和Omp N,以及一种功能未知的蛋白。利用同源克隆,成功地从ML01株基因组中扩增到了P42、P36、P31的基因。序列测定和比对结果显示,ML01株的这3个基因与哈维氏弧菌ATCC 33843(Gen Bank CP009467)的相应基因相比,其开放阅读框(Open reading frame,ORF)序列的相似性分别为97.08%、100%、99.67%,其编码的多肽序列的相似性分别为99.71%、100%和99.93%。本研究对进一步分析哈维氏弧菌ML01株的致病机理、研发该菌的亚单位疫苗具有重要的参考价值。

Pathogenic bacterium Vibrio harveyi: an endosymbiont in the marine parasitic ciliate protozoan Cryptocaryon irritans

Vibrio harveyi, known as a pathogenic bacterium caused severe secondary bacterial infections of the large yellow croaker Larimichthys crocea, was identified as an endosymbiont in the marine parasitic ciliate protozoan Cryptocaryon irritans. Meta 16 S sequencing method was used to identify the bacterial flora in C. irritans, and V.harveyi was isolated via culture-dependent method. Vibrio harveyi was observed in cytoplasm of C. irritans at the stage of tomont both by transmission electron microscopy and by Fluorescence in situ hybridization; no signal,however, was detected in nucleus area. The relationship between V. harveyi and C. irritans and the role of endosymbiotic V. harveyi in C. irritans merit further investigation.

Inhibitory Activity of Prunus mume,Copitis chinensis and Crataegus pinnatifida on Vibrio harveyi and Its Biofilm

[Objective] This study was conducted to determine the in-vitro inhibitory effects of Prunus mume,Coptis chinensis and Crataegus pinnatifida on Vibrio harveyi and its biofilm. [Method]The inhibitory zone diameters of the three Chinese herbal medicines against V. harveyi were determined by agar diffusion method; the minimal inhibitory concentration( MIC) and minimal bactericidal concentration( MBC) values of the three Chinese herbal medicines against V. harveyi were determined by doubling dilution method; and the effects of the three Chinese herbal medicines on the formation of V. harveyi biofilm were determined by methyl thiazolyl tetrazolium( MTT) method. [Result]The three Chinese herbal medicines all inhibited V. harveyi to different degrees. C. chinensis and C. pinnatifida and P. mume exhibited the inhibitory zone diameters of( 17. 62 ± 0. 04),( 20. 16 ± 0. 08) and( 30. 76 ± 0. 26) mm against V. harveyi,respectively. P. mume and C. pinnatifida had strong inhibitory effects on V. harveyi. The MIC and MBC values of P. mume against V. harveyi were 7. 812 5 mg/ml; the MIC and MBC values of C. pinnatifida against V. harveyi were 31. 25 mg/ml; and the MIC and MBC values of C. chinensis against V. harveyi were 62. 5 mg/ml. P. mume had the strongest antibacterial and bactericidal ability. The MIC values of C. pinnatifida,C. chinensis and P. mume against V. harveyi were 7. 81,7. 81 and 1. 96 mg/ml,respectively,i. e.,P. mume exhibited the lowest MIC. [Conclusion] P. mume,C. pinnatifida and C. chinensis all have inhibitory effects on V. harveyi and its biofilm,and P. mume has the strongest bactericidal ability.

Host gut-derived Bacillus probiotics supplementation improves growth performance, serum and liver immunity, gut health, and resistive capacity against Vibrio harveyi infection in hybrid grouper(♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatus)

Several reports have revealed the vital role that probiotics play in fish growth and health.However,few works are available for host gut-derived probiotics on the growth,immunity,and gut microbiota of fish,especially in hybrid grouper (♀Epinephelus fuscoguttatus×♂Epinephelus lanceolatus) due to their isolation difficulty and functional verification.This study aimed at assessing 3 host gut-derived Bacillus species?effects on the growth,immune and antioxidant-biochemical responses,haematological parameters,intestinal morphology,immune-related gene expression,gut microbiota,and disease resistance against Vibrio harveyi in hybrid grouper.A total of 480 hybrid grouper (initial weight=9.03±0.02 g) were randomly allotted into 4 groups,namely,the group fed a basal diet without probiotic inclusion (control,B0),the group fed the basal diet with Bacillus velezensis GPSAK4 (BV),the group fed the basal diet with Bacillus subtilis GPSAK9 (BS),and the group fed the basal diet with Bacillus tequilensis GPSAK2 (BT) strains at 1.0×10^(9)CFU/g.After a 6-week feeding trial,the results revealed significant improvements (P<0.05) in the growth performance,whole fish-body proximate composition,blood haematological parameters,serum,liver,and intestinal biochemical indexes,intestinal morphology,and protection against V.harveyi pathogen in the probiotic-treated groups compared with the untreated.Additionally,the expressions of intestinal tight junction genes (occludin and ZO1),pro-and anti-inflammatory genes,including IL1β,IL6,IL8,TNFa,MyD88,IL10,and TGFβ,were upregulated (P<0.05) after Bacillus species administration.Host gut-derived Bacillus supplementation shaped the gut microbiota by significantly increasing (P<0.05) the relative abundance of Proteobacteria,Bacteroidetes,Actinobacteria (except the BS group),Acidobacteria(except the BT group),Cyanobacteria (except the BV and BT groups),and Verrucomicrobia phyla,as well as known beneficial genera (Romboutsia,Turicibacter,Epulopiscium,Clostridium_sensu_stricto 1 and 13,Lactobacillus,and Bacillus),but significantly decreased (P<0.05) the abundance of Firmicutes,Chloroflexi,and Fusobacteria phyla,and purported pathogenic genera (Staphylococcus and Photobacterium) compared with the control group.Collectively,the results suggest that B.velezensis GPSAK4,B.subtilis GPSAK9(especially this strain),B.tequilensis GPSAK2 dietary supplementation at 1.0×10^(9)CFU/g has positive effects on the intestinal health of hybrid grouper via microbial composition modulation,thus enhancing the assimilation and absorption of nutrients to boost fish growth,immunity,and disease resistance.

4株长牡蛎(Crassostrea gigas)致病性哈维氏弧菌(Vibrio harveyi)鉴定及其毒力基因检测

为明确江苏连云港养殖长牡蛎大规模死亡病因,对分离自长牡蛎的4株病原菌开展了致病性、表型特征、分子特征等研究,结果表明4株病原菌均具致病性,4株病原菌ML1、ML2、ML3、ML4对长牡蛎的半数致死量LD50分别为2.8×105、1.1×105、1.8×105和1.6×105 CFU/mL;4株病原菌均为呈短杆状革兰氏阴性细菌,氧化酶阳性,还原硝酸盐,能利用甘露醇、纤维二糖,形态及理化特性结果表明4株分离菌与弧菌属的哈维氏弧菌亲缘关系最近;16SrRNA、gyrB、rpoA基因同源性检索及系统发育学分析结果表明4株分离菌与GenBank数据库中哈维氏弧菌的基因序列相似性最高且在系统发育树中聚为1个分支;根据4株分离菌的致病性、表型与分子特征,表明引起长牡蛎大规模死亡的病原为哈维氏弧菌。为进一步明确分离菌株毒力基因的携带情况,检测了分离鉴定的4株病原菌的9种毒力基因,结果表明,4株病原菌均可检测到luxR、toxR、vhhA和vhhB 4种毒力基因,扩增片段大小分别为679bp、390bp、1324bp和216bp,其他5种毒力基因未检测到,且分离鉴定的4株病原哈维氏弧菌携带相同的毒力基因,这些毒力基因可作为检测致病性哈维氏弧菌的分子标记。

Antigenicity analysis of Vibrio harveyi TS-628 strain

Vibrio harveyi,the major causative agent of vib-riosis,affects a diverse range of marine cultured organisms over a wide geographical area.However,reports about screening the effective antigen and research on vaccines of V.harveyi are scarce.Flagellin,lipopolysaccharide(LPS)and outer membrane proteins(OMP)are major immunogenic antigens in many Gram-negative bacteria.In this study,the flagellin,OMP and LPS of the V.harveyi TS-628 strain iso-lated from infected groupers were extracted and Western blot analysis was used to detect the antigenicity of these extrac-tions.Results of the Western blot assay reveal that there are four positive flagellin bands:35 kDa,38 kDa,43 kDa,and 52 kDa,of which the 43 kDa and 52 kDa bands displayed the strongest positive reaction.There are five positive OMPbands about 35 kDa,38 kDa,43 kDa,47 kDa,and 52 kDa,of which the 43 kDa appeared to have the strongest positive reaction although the other four proteins also displayed strong reac-tions.However,LPS is Western blot-negative.These results indicate that the 43 kDa and 52 kDa flagellin and OMP of size 43 kDa,52 kDa can be candidates for developing vaccines against V.harveyi.

In vitro biofilm forming capacity on abiotic contact surfaces by outbreak-associated Vibrio harveyi strains

Objective:To evaluate the in vitro biofilm forming capacity on abiotic food contact surfaces by Vibrio harveyi(V.harveyi)strains.Methods:Thirty six Gram-negative V.harveyi strains were isolated from various street vended seafood outlets in a food processing line and evaluated for their ability to produce mucoid biofilms on food contact surfaces using a microplate assay.Phenotypic characterization of mucoid biofilm producing V.harveyi strains were screened on Congo red agar,thiosulfate-citrate-bile salts-sucrose agar and tryptic soy agar,respectively.Results:Only five V.harveyi strains(14%)were mucoid biofilm producers characterized by formation of black colonies,whereas the remaining 31 strains(86%)were not capable of producing biofilm characterized by formation of red colonies or pinkish-red colonies with darkening at the centre.The morphological,physiological and biochemical characteristics of these isolates were studied using standard protocols.Strain identification was confirmed by polymerase chain reaction targeted to species-specific polymerase chain reaction primers VH-1 and VH-2 corresponding to variable regions of V.harveyi 16S rRNA sequence.All the biofilm-forming strains showed resistance to at least three antimicrobial compounds tested.V.harveyi strains isolated from various seafood were able to form biofilms of different capacity,and the strains VB267,VB238 and VB166 isolated from cat fish,shrimp and eel fish exhibited significantly greater biofilm forming ability compared to other isolates.Conclusions:It can be concluded from the present study that the strain VB166 was able to better attach and form subsequent biofilms on glass and stainless steel compared to high density polyethylene.These properties allow these bacteria to survive,proliferate and persist in street vended seafood outlets.

哈氏弧菌(Vibrio harveyi)recA基因的克隆与原核表达

SOS反应不仅能够促使细菌本身产生耐药性,而且与细菌耐药基因的水平转移密切相关。rec A是细菌SOS反应的重要基因,影响SOS反应的开启和关闭。本研究以水产养殖经济动物的条件致病菌哈氏弧菌ZJ0603基因组DNA为模板,设计同源引物克隆出SOS基因rec A（recombinase A）,并对rec A基因进行了生物信息学分析。并构建了rec A基因的原核表达载体PGEX-rec A,在大肠杆菌BL21（DE3）中成功诱导表达,对Rec A蛋白的诱导温度、时间、IPTG浓度等条件进行了优化。结果表明,rec A基因的开放阅读框为693 bp,编码230个氨基酸,Rec A蛋白理论分子量为25.29 k D。重组蛋白表达的优化条件为37℃、0.04 mmol/L IPTG诱导6 h。

哈氏弧菌(Vibrio harveyi)lexA基因的克隆与原核表达

LexA是调节SOS反应的阻遏蛋白,而且与细菌耐药性的形成密切相关。本研究以水产养殖经济动物的条件致病菌哈氏弧菌ZJ0603基因组DNA为模板,设计同源引物克隆出SOS基因lexA,并对lexA基因进行了生物信息学分析;构建了lexA基因的原核表达载体PGEX-lexA,在大肠杆菌BL21（DE3）中诱导表达成功,还对LexA蛋白表达的诱导温度、时间、IPTG浓度等条件进行了优化。结果表明,lexA基因的开放阅读框为621 bp,编码206个氨基酸,LexA蛋白理论分子量为22.68 kD;重组蛋白表达的最优条件为37℃、0.04 mmol/L IPTG诱导6 h。