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1.
We cloned and sequenced a prtV-like gene from Vibrio anguillarum M3 strain.This prtV gene encodes a putative protein of 918 amino acids,and is highly homologous to the V.cholerae prtV gene.We found that a prtV insertion mutant strain displayed lower gelatinase activity on gelatin agar,lower protease activity against azocasein,and lower activity for four glycosidases.This prtV mutant strain also had increased activity for two esterases in its extracellular products,as analyzed by the API ZYM system.In additi...  相似文献   

2.
Mutant J61321 with enhanced siderophore production of Alteromonas aurantia AI8 was obtained after a series of chemical-physical mutageneses. It was found that the antibacterial activity against Vibrio anguillarum W-1 and siderophore production of the mutant were higher than those by the original strain A 18 which had been used in mariculture. The results of the specific assay（Csaky and Arnow methods） of siderophore showed that the sidrophore with hydroxamate group was produced by mutant J61321 and the original strain A 18, respectively, while the siderophore with catechol group was yielded by strain W-1 （Aibrio anguillarum）. Meanwhile, the siderophore yield, antibacterial activity and anti-chelator activity of strain J61321 were higher than those of its parent strain A 18.  相似文献   

3.
A Gram negative bacterium Ar/W/b/75°25＇N/1 producing extracellular alkaline protease was isolated from surface water of latitude 75°25＇N, and longitude 162°25＇W in Chukchi sea, Arctic. The strain can grow at the temperature range from 7℃ to 30℃, and grow better at 30(℃. It can not grow at 40℃. Keeping certain salinity concentration in medium is necessary for cell growth. It grows well in medium containing salinity concentration from 0. 5 % to 10 % sodium chloride. Glucose, sucrose and soluble starch can be utilized by the strain, among which glucose is the optimal carbon source. Peptone is the optimal organic nitrogen source for cell growth and protease producing, and ammonium nitrate is the optimal inorganic nitrogen source.About 75.7% of total protease of the strain are extracellular enzyme. Optimal temperature for proteolytic activity is at 40℃. Protease of the strain keeps stable below 40℃, and shows high proteolytic activity within the pH range from 7 to 11.  相似文献   

4.
Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming.The rapid detection and identification of V.anguillarum,and other pathogens that infect marine organisms,is crucial to effective disease management.In this study,we developed a loop-mediated amplification (LAMP) assay to detect V.anguillarum in an hour in a single tube without the need for thermal cycling.Conserved regions of the metalloproteinase (empA) gene of V.anguillarum served as the targets for primer design.A fragment of the empA gene was amplified at 65°C in the presence of the primer mixture and Bst DNA polymerase.In the optimized LAMP assay,6.7 pg of V.anguillarum DNA could be detected.Six strains of V.anguillarum and 17 strains of non-V.anguillarum bacteria were used in this study to evaluate the species specificity of the primers.The six V.anguillarum strains gave a positive result in the LAMP assay.This method was also validated in V.anguillarum-infected fish.This LAMP method is more sensitive than PCR in the detection of V.anguillarum and shows good species specificity.The LAMP assay is therefore an effective method for the quick detection of V.anguillarum both in the laboratory and in the field.  相似文献   

5.
Interleukin-17(IL-17), a prototype member of IL-17 family, plays an important role in defending against extracellular pathogens as a pro-inflammatory cytokine. The function and distribution of IL-17 have been extensively studied in many vertebrates. However, few study has focused on the role of IL-17 in invertebrates, especially in mollusks. In this study, an IL-17 homolog was identified in Octopus ocellatus, which was designated as OoIL-17. The phylogenetic analysis showed that OoIL-17 is clustered well with other invertebrate IL-17, indicating it is highly similar with the IL-17 of other invertebrates. The expression of OoIL-17 gene was analyzed with qRT-PCR in a variety of healthy tissues and the hemocytes infected with Vibro anguillarum or Micrococcus luteus. The mRNA of OoIL-17 gene is constitutively expressed at different levels in all examined tissues of healthy O. ocellatus, including mantle, stomach, hemocytes, muscle, gonad, hepatopancreas, systemic heart and gill. The lowest expression was observed in mantle while the highest was observed in hepatopancreas. The expression level of OoIL-17 gene is significantly up-regulated in O. ocellatus hemocytes upon infection with V. anguillarum and M. luteus, indicating its active involvement in the host immune response against bacterial pathogens. The results laid the foundation for further understanding the innate immune mechanisms of IL-17 in O. ocellatus and mollusks.  相似文献   

6.
Freeze-drying, continuous passage and ultra-low temperature cryopreservation are often used to preserve pathogens. In this study, Vibrio anguillarum was rejuvenated by intramuscular infection as the initial strain. The difference between cells preserved with different preservation methods and their initial strains were compared with physiological and biochemical methods and through antibiotics resistance analysis. The composition of protectants for freeze-drying V. anguillarum was optimized. We found that the optimal composition of protectants was 8% of trehalose, 12% of skim milk, 8.0% of lactose, 2.0% of sodium citrate, 12.0% of serum and 8.0% of mannitol. The indexes of lysine decarboxylase and urease changed after continuous passage. The urease reaction changed after freeze-drying and freeze-thawing, but the reaction can be restored to the initial after freeze-drying. Based on the antibiotics resistance analyses, the sensitivity of V. anguillarum to different drugs including rifampicin, erythrocin, furazolidone, ceftazidime, lomefloxacin, gentamycin, azithromycin, doxycycline, ampicillin, co-trimoxazole and cefoperazone changed after different treatments, and some of these changes can be restored to the original through activation culture. In sum, compared with cryopreservation and continuous passage, the freeze-drying is more sustainable for the long-term preservation of V. anguillarum, which showed a better effect in maintaining the original characteristics of pathogen.  相似文献   

7.
We cloned and sequenced a prtV-like gene from Vibrio anguillarum M3 strain. This prtV gene encodes a putative protein of 918 amino acids, and is highly homologous to the V. cholerae prtV gene. We found that a prtV insertion mutant strain displayed lower gelatinase activity on gelatin agar, lower protease activity against azocasein, and lower activity for four glycosidases. This prtV mutant strain also had increased activity for two esterases in its extracellular products, as analyzed by the API ZYM system. In addition, the prtV mutant strain exhibited decreased growth in turbot intestinal mucus and reduced hemolytic activity on turbot erythrocytes. Infection experiments showed that the LD50 of the prtV mutant strain increased by at least 1 log compared to the wild-type in turbot fish. We propose that prtV plays an important role in the pathogenesis of V. anguillarum.  相似文献   

8.
Vibrio anguillarum is a common bacterial pathogen in fish.However,little is known about its pathogenic mechanism,in part,because the entire genome has not been completely sequenced.We constructed a fosmid library for V.anguillarum containing 960 clones with an average insert size of 37.7 kb and 8.6-fold genome coverage.We characterized the library by end-sequencing 50 randomly selected clones.This generated 93 sequences with a total length of 57 485 bp covering 1.4% of the whole genome.Of these sequences,58...  相似文献   

9.
Bacteria-infected turbots Scophthalmus maximus with septicaemia were examined between 2001 and 2004 in aspects of the conditions of disease occurrence, clinical syndromes and pathological changes. The phenotypic information of pathogenic bacteria was studied, including morphology, physiological and biochemical characteristics, and the mol% G C of the DNA. In addition, representative strains (S010623-1, LH031120-1) were selected for molecular identification by partial 16S rRNA gene sequencing. The results show that the isolates (LH031120-1 to LH031120-6, HT040308-1 to HT040308-6, HT040620-1 to HT040620-6) from three farms were identified as Edwardsiella tarda. The isolates (S010610-1 to S010610-10, S010623-1 to S010623-20) from one farm were identified as Listonella anguillarum. We conducted studies on the pathogenicity of isolates by artificial infection, and revealed all infected groups in morbidity and mortality. The septicaemia infected turbot showed a syndrome similar to that of the naturally infected fish. Antibiotic sensitivity showed that of 37 antimicrobial agents, E. tarda was sensitive to 27 agents, and L. anguillarum was sensitive to 21 agents.  相似文献   

10.
Studies were conducted to determine the cause of the acute mortality of half-smooth tongue sole Cynoglossus semilaevis Günther juveniles in a fish farm in Jimo, Shandong Province, China, in June 2006. Gross signs of the diseased tongue sole included several petechiae and ecchymoses on the body and fin necrosis and hemorrhagic lesion at the base of the fin. Bacteria were isolated from kidney, liver and hemorrhagic lesions of the diseased tongue sole. Among14 strains, SJ060621 was proved to be highly virulent to juvenile tongue sole with LD50 value of 1.0×105 colony forming units (CFU)mL-1, while the remaining 13 were avirulent. Among the 16 antibiotics tested, SJ060621 was sensitive to gentamicin and nitrofurantoin. It was identified as Listonella anguillarum with conventional plate and tube tests in combination with API 20E analysis. 16S rRNA gene and partial HSP60 gene sequenceing analysis revealed that the strain was highly homologous with L. anguillarum. Examination of the infected musculature by electron microscopy indicated numerous bacteria and lots of macrophages containing phagocytosed bacteria. Histopathological investigations revealed severe necrotic degenerative changes in the infected organs. Indirect immunofluorescence assay (IFA) was employed to detect the location of occurrence of bacteria, and bacteria were found in aggregations in the inflammatory areas in musculature.  相似文献   

11.
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12.
107 strains producing protease were screened from 260 strains of Antarctic psychrophilic bacteria, among which proteolytic activity of five strains was more than 45 U ml^-1. The 16S rRNA gcne sequences homology and phylogcnetic analysis of five Antarctic psychrophillc bacteria showed that NJ276, NJS-9, NJ16-70,NJ345 belonged tO the described genus Pseudoalteromonas and NJ341 belonged to the genus Colwellia. The growth and the protease characteristic of four Antarctic psychrophilic bacteria had been studied, and the result showed that the 6ptimal temperature for growth and protease-produeing of four strains was about 10℃. Their growth and protease-produeing were still high during incubatlng 2-5 days. The maximum proteolytic activity occurred at pH 9 for four Antarctic psychrophilic bacteria. The optimal temperature of protease action of both strains NJ276 and NJ5-9 was about 50℃, however, the optimal temperature of protease aetlon of both strains NJ341 and NJ345 was about 40 ℃, and their proteolytic activity under 0℃ exhibited nearly 30% of the maximum activity, but their thermal stabilities were weaker. These results indicated that proteases from NJ341 and NJ345 were low-temperature proteases.  相似文献   

13.
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14.
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 th...  相似文献   

15.
An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates （azocasein and casein）. The specific protease activity was 17.15 folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55℃ and the optimal pH value was 5.5. Ion Ca^2＋ could enhance the proteolytic activity of the protease, while Cu^2＋, EDTA and PMSF could inhibit its activity.  相似文献   

16.
抗菌肽对凡纳滨对虾生长和机体免疫的影响   总被引：1，自引：0，他引：1  
基础饲料(对照组)中添加10g/kg抗菌肽制剂喂养体重0.10±0.01g、体长2.17±0.11cm的凡纳滨对虾(Penaeus vannamei)30d,进行10d的水浴攻毒(鳗弧菌10~8/mL),探讨饲料中添加抗菌肽对凡纳滨虾生长、成活率、免疫保护率,以及肌肉中溶菌酶活性、超氧化物歧化酶活性、总抗氧化能力的影响。结果表明:饲料中添加抗菌肽可显著提高凡纳滨对虾相对增重率、成活率和对致病菌的免疫保护率(P<0.05),但对饲料系数无显著影响(P>0.05);未受外界微生物刺激时,饲料中添加抗菌肽对凡纳滨对虾总抗氧化能力有促进作用(P<0.05),受外界微生物刺激后,饲料中添加抗菌肽喂养凡纳滨对虾可提高对虾超氧化物歧化酶活性(P<0.05)和总抗氧化能力(P<0.01)。因此饲料中添加抗菌肽喂养凡纳滨对虾可提高其机体非特异性免疫。  相似文献   

17.

A new chemically mutagenic mutant ofSynechococcus PCC7942 named high-CO2 requiring mutant, which could grow at 4% CO2 but could not grow at air levels of CO2, was isolated. Comparative studies on some physiological aspects of the mutant and high-CO2 growing cells (growing at 4% CO2) were conducted. The result showed that the mutant had lower growing rate, about 1/40th photosynthetic affinity to inorganic carbon, 25% lower carbonic anhydrase (CA) activity, lower quenching rate of chlorophyll fluorescence, and about 1/2 alkalinization rate of the medium. The CA activity responses of the two types of cells to different concentration of CO2 were determined. Upon the addition, of inorganic carbon (Ci), the rate of active Ci uptake described by the rate of chlorophyll fluorescence quenching of the mutant was obviously lower compared with that of the high-CO2 growing cells; the size of the internal inorganic carbon pool size detemined by the extent of fluorescence quenching of the mutant was also smaller.

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18.
An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates (azocasein and casein). The specific protease activity was 17.15 folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2 kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55°C and the optimal pH value was 5.5. Ion Ca2+ could enhance the proteolytic activity of the protease, while Cu2+, EDTA and PMSF could inhibit its activity.  相似文献   

19.
1　INTRODUCTIONThepenaeidshrimpcultureindustriesintheworlddevelopedfromtheirexperimentalbeginningslessthanthirtyyearsagointoamajoraquaculturein dustrytodaythatprovideshundredsofthousandsofjobs,billionsofU .S.dollarsinrevenue,andaug mentstheglobalfoodsupplywithahighvaluecrop(LighterandRedmam ,1 998) .Upto 1 995,farmedshrimpatvolumeof71 2 0 0 0MT ,almostallconsistingofPenaeidspecies,accountedfor 2 7%ofworldshrimpproduction.Inthatyear,asoneofthemostimportantculturedshrimp,theproductionofP…  相似文献   

20.
We investigated the effects of lipopolysaccharide(LPS) and dopamine(DA) on the activation of the prophenoloxidase(proPO) system of Litopenaeus vannamei.LPS and DA were shown with a negative dose-dependent effect on hyalne cells(HC),semi-granular cells(SGC),large granular cells(LGC),and total haemocyte count(THC).When haemocytes were treated with LPS or DA,serine proteinase activity and intracellular phenoloxidase(PO) activity were significantly reduced,but extracellular PO activity increased significantly.These findings indicated that the reduction in haemocyte counts was mainly because of the degranulation and activation of the proPO system from semi-granule and large granule cells.The PKC inhibitor,chelerythrine,and the TPK inhibitor,genistein,had an inhibitory effect on extracellular PO activity,while serine proteinase and intracellular PO activity increased.This suggests that the LPS and DA induce the activation of proPO in haemocytes via PKC and TPK-related signaling pathways,but serine proteinase may be activated only by PKC,as the genistein effects were not statistically significant.Electrophoresis analysis revealed that POs induced by LPS or DA have the same molecular mass and high diphenolase activity.Two PO bands at 526 kDa and 272 kDa were observed in PAGE,while in the haemocyte lysate supernatant(HLS),only a 272-kDa band was observed.This band was resolved after SDS-PAGE under non-reducing and reducing conditions into two groups of POs,166 kDa and 126 kDa,and 78.1 kDa and 73.6 kDa,respectively,suggesting that PO in L.vannamei is an oligomer,which may have different compositions intra-and extracellularly.  相似文献   

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