海南地区溶藻弧菌遗传多样性的RAPD分析

以来源于泰国的菌株TG06003作对照,对采自海南地区的27株溶藻孤菌（Vibrio alginolyticus）环境菌株进行了随机扩增多态性DNA标记（RAPD）分析,其中6株为毒力菌株,另外22株为非毒力菌株。实验共得到70个多态性条带,多态性比率（PP13）为98．6％。基因多样度的算术平均数和加权平均数分别为0...     

A VIBRIO ANGUILLARUM STRAIN ASSOCIATED WITH SKIN ULCER ON CULTURED FLOUNDER, PARALICHTHYS OLIVACEUS

ＩＮＴＲＯＤＵＣＴＩＯＮＩｎｃｒｅａｓｉｎｇａｔｔｅｎｔｉｏｎｗａｓｆｏｃｕｓｅｄｏｎｔｈｅｎｅｅｄｔｏｓｔｕｄｙｔｈｅｂａｃｔｅｒｉｏｌｏｇｙｏｆｍａｒｉｎｅｆｉｓｈｉｎＣｈｉｎａ ,ｂｅｃａｕｓｅｏｆｂａｃｔｅｒｉａａｓｓｏｃｉａｔｉｏｎｗｉｔｈｈｉｇｈｍｏｒｔａｌｉｔｙｉｎｍａｒｉｎｅｆｉｓｈｃｕｌｔｕｒｅａｎｄｃｏｎｓｅｑｕｅｎｔｅｃｏｎｏｍｉｃｌｏｓｓｔｏｆｉｓｈｆａｒｍｓ (Ｄｅｎｇｅｔａｌ.,1 996;Ｌｉｎｅｔａｌ.,1 999) .Ｖｉｂｒｉｏｇｅｎｕｓｂａｃｔｅｒｉａｈａｖｅｅｍｅｒｇｅｄａｓｔ…     

A VIBRIO ANGUILIARUM STRAIN ASSOCIATED WITH SKIN ULCER ON CULTURED FLOUNDER, PARALICHTHYS OLIVACEUS

The characteristics of a bacterium strain M3, isolated from cultured flounderParalichthys olivaceus with remarkable external sign of skin ulcer during an epizootic outbreak, indicated that the bacterium belonged to the speciesVibrio anguillarum. Challenge by I.M. (intramuscular injection), bath, and oral administration with M3 showed that it was highly pathogenic forParalichthys olivacues. The LD₅₀ dose was 5.144×10³ CFU/ per fish infection by I.M. injection. Recovered inoculated bacteria from the surviving fish revealed that the asymptomatic carriers could be a latent contagious source. Study of the effect of bacterial culture CFS (cell-free-supernatant) showed that the exotoxins produced by M3 play an important role in its pathogenicity for flounder. The resistance of M3 to 36 out of 41 antibiotics indicated that the bacterial disease outbreak was mainly attributable to the frequent and excessive use of antimicrobial agents; and that vaccination would be an effective precaution against bacterial disease. Contribution No. 4107 from Institute of Oceanology, Chinese Academy of Science. Contribution No. 270 from Experimental marine Biology Laboratory, CAS. Funded by projects under the Major State Basic Research Development Program, Grant G1999012003.     

Interaction of Haematopoietic Tissue Cultures of the Dublin Bay Prawn,Nephrops Norvegicus(L.),with the Causal Agent of Luminous Vibriosis Vibrio Harveyi

Vibrio harveyi cells (dose = ＞ 103 cells mL 1) and extracellular products (ECP; ＞25μg m L-1 of total protein concentration) destroyed haematopoietic cultures of Nephrops norvegicus within 24 h of exposure. Cytopathic effects (CPE)started after 4 h of exposure to the bacterial cells, with some granularity in the cytoplasm, mostly in cells in the outer periphery of the explant growth. At the end of the infection, a considerable number of nuclei remained attached to the substrate,apparently unaffected. Following exposure to ECP, initial deterioration was observed at 2 h with the presence of granularity in the cytoplasm of＜ 20% cells, and few cells displayed small vacuoles around the nuclei. Parallel results were obtained using whole animal experiments, with V. harveyi cells being lethal to nephrops within 24 h.     

溶藻弧菌HY9901血红素结合蛋白HutB的原核表达及纯化

为探究溶藻弧菌(Vibrio alginolyticus)HY9901血红素结合蛋白HutB作为疫苗候选抗原的可能性,采用PCR方法扩增V.alginolyticus HY9901 hutB基因全长序列,克隆到pMD18-T载体,经双酶切、连接后,定向插入到pET-28a(+)中,构建原核表达载体pET-HutB,转入大肠杆菌BL21(DE3)中进行IPTG诱导表达。SDS-PAGE验证后,优化诱导表达条件和纯化条件,并进行Western-blot分析。结果表明,HutB蛋白在E.coli中诱导表达的最优条件:0.4 mmol/L IPTG,37℃诱导4h,表达的蛋白分子量与预期大小相符,主要以包涵体的形式存在,300 mmol/L咪唑洗脱时效果最佳,能与鼠抗His-tag单抗特异性结合。     

一株溶藻弧菌噬菌体的生理特性研究

对从湛江市水产品批发市场分离得到一株溶藻弧菌噬菌体的生理特性进行测定,结果表明:该株噬菌体噬菌斑12 h直径为1～1.5 mm,对紫外线敏感,暴露在紫外灯(20 W,30 cm)下30 min可丧失全部裂解活性;在pH8～11范围内裂解活性较强,最适pH值为11,裂解周期为65 min,对65℃以上的高温敏感,对乙醚和氯仿有较好的耐受性,对抗病毒药物阿昔洛韦敏感,最大常规稀释度为106CFU/mL,最佳感染复数为10。     

泥鳅(Misgurnus anguillicaudatus)病原霍乱弧菌(Vibrio cholerae)PCR 与LAMP检测方法的比较研究

采用分子生物学方法,以霍乱弧菌lolB为靶基因设计特异性引物,进行了霍乱弧菌的PCR和环介导等温核酸扩增(Loop-mediated isothermal amplification,LAMP)检测技术研究,并对它们的特异性、灵敏性和实际应用进行了比较.结果表明,所建立的PCR检测霍乱弧菌的方法最低检测限为4.0× 103 CFU/ml; LAMP检测方法在65℃下恒温扩增60min,检测限为4.0×101 CFU/ml,反应产物加入荧光染料SYBR Green Ⅰ后反应液呈现明显的绿色;以温和气单胞菌、副溶血弧菌、鳗弧菌及美人鱼弧菌为对照菌株,检测结果均为阴性;霍乱弧菌人工染菌的8种水产品进行PCR及LAMP检测,结果均为阳性,而未染菌组均为阴性;PCR及LAMP检测霍乱弧菌的方法均具有灵敏度较高、特异性强等优点,且LAMP检测霍乱弧菌的方法灵敏度是PCR方法的100倍,更适合于养殖现场检测的推广使用.     

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-Tn10/Kan transposon-induced mutants with deficient chemotactic motility were constructed, screened, and identified. Sequence analysis revealed that the 465-bp fragment (GenBank accession number HM630274) flanking the transposon insertion site in mutant TS-CM1 had the highest identity (96.9%) with a hypothetical protein gene of V. harveyi ATCC 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 protein 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, adhesion, and infectivity of themutants showed that disruption of either the flagellin gene or energymetabolism gene led to subsequent loss of chemotactic motility and changes in growth, motility, adhesion, and virulence of the pathogenic V. harveyi. Hence, the flagellin gene and crucial energy metabolism gene played an important role in the chemotacticmotility of V. harveyi.     

中国对虾血清中酚氧化酶活力研究

１９９５年７－９月,以养殖中国对虾为材料,实验室用弧菌菌悬液注射感染对虾,血甭中测得一定量的酚氧化酶（ＰＯ）。正常状态下对虾血清中ＰＯ活力变化不大,而濒死对虾血清中ＰＯ活力变化较大。较低浓度的菌悬液能有效地提高ＰＯ活力,促使有机体对病原体的防御能力,这在实践中有重要意义。