微生物能力验证样品制备工艺及水产品企业水平测试结果评价

为了提高舟山市水产品企业实验室微生物检测能力,保证企业自检结果准确可靠,根据舟山市进出口检验检疫工作的特点,选取了水产品中3个常规的微生物检测项目,即菌落总数、副溶血弧菌和金黄色葡萄球菌检测,采用了真空冷冻干燥技术制备一系列微生物能力验证样品,对舟山市88家水产品企业的实验室进行微生物能力验证。结果表明采用本工艺制备的能力验证样品具有稳定性好、贮存方便、保存期长等优点。从水产品企业微生物水平测试结果来看,菌落总数项目的满意率为81.8%;副溶血弧菌检测的正确率为93.8%,而金黄色葡萄球菌检测的正确率为82.1%。检测结果存在偏差的主要原因是检测中未使用标准菌株进行参照、试剂在使用前无进行质量核查、对某些生化反应判断失误等。     

黄连水提物对卵形鲳鲹源溶藻弧菌的抑菌作用

卵形鲳鲹(Trachinotus ovatus)是一种生长快速且肉质鲜美的经济型海水养殖鱼类,但是近些年为满足人们的日常需求,在不断扩大养殖的同时病菌也逐渐泛滥。溶藻弧菌(Vibrio alginolyticus)是华南沿海地区海水养殖鱼类细菌性鱼病的主要致病菌之一,给水产养殖业带来巨大损失。本研究对黄连(Coptis chinensis Franch)的抗菌作用进行系统研究,目的是为由溶藻弧菌引起的爆发性细菌性鱼病的高效防治提供科学的用药依据。本研究采用二倍稀释法测定黄连水提物对卵形鲳鲹源溶藻弧菌的最低抑菌浓度(MIC)、最低杀菌浓度(MBC)和半数致死量(LD50),并使用BCA蛋白浓度测定试剂盒测定细菌超声破碎上清液中可溶性蛋白含量。结果表明,黄连水提物对溶藻弧菌具有明显的抑制作用,其对溶藻弧菌的MIC、MBC和LD50的值分别为7.800mg/mL、31.250mg/mL和15.625mg/mL。胞内可溶性蛋白含量变化的结果提示:黄连发挥抑菌作用的机制可能是通过损伤细菌的细胞壁导致细菌内容物的释放并引起菌体裂解死亡。黄连具有发展成为一种高效抗水产病害中草药制剂的潜力。     

费氏弧菌检测土壤联合毒性模型分析

在获得费氏弧菌(Vibrio fischer)相对发光强度与重金属离子浓度关系的基础上,采用响应面分析法建立相对发光强度与土壤联合毒性的数学模型.同时,对五处正在从事生产的工厂土壤进行采样检测验证模型的可行性.结果表明:通过建立的模型成功得出五处采样地点的土壤状况.其中,五处采样点中浅层土生物毒性均大于深层土,同一工厂排污口和生产车间之间土壤生物毒性差异较小;各采样点土样中的离子联合毒性均位于模型中的Ⅱ级毒性区间,相当于重金属离子含量为0.01 mg·L^-1.说明,该模型可用于初步判断土壤生物毒性.     

海洋蛭弧菌BDSG1的发酵培养条件研究

从厦门市集美鳌园附近海域分离得到一株海洋蛭弧菌BDSG1.实验结果表明,该菌的最适生长温度为25℃,最适pH值为8.3.在人工生态环境中对该蛭弧菌与宿主弧菌相互作用的消长情况的研究表明,蛭弧菌BDSG1对弧菌有较强的裂解和净化作用.通过单因素及五因子正交试验对蛭弧菌BDSG1发酵生产条件进行优化,得到了最优培养基配方,发酵最佳时间为50 h,菌液浓度可达9.5×1010pfu/mL.     

Overexpression and export of Vibrio anguillarum metalloprotease in Escherichia coli

Vibrio anguillarum metalloprotease, an extracellular zinc metalloprotease involved in the virulence mechanism of Vibrio anguillarum, is synthesized from the empA gene as a 611-residue precursor and naturally secreted via Sec secretion pathway in Vibrio anguillarum. In this study, heterologous expression of the empA gene encoding metalloprotease and export of the recombinant metalloprotease in Escherichia coli were examined. The empA gene was subcloned into pBAD24 with arabinose promoter and sequenced. The sequence encoded a polypeptide (611 amino acids) consisting of four domains: a signal peptide, an N-terminal propeptide, a mature region and a C-terminal propeptide. The empA gene inserted in plasmid pBAD24 was overexpressed in TOP10 strain of E. coli after arabinose induction. The 36kDa polypeptide of the recombinant metalloprotease as the mature protease was further confirmed by SDS-PAGE and immunoblotting. It was found that recombinant metalloprotease with the EmpA activity and antigenicity was exported into the periplasm of Escherichia coli cells via Sec translocation pathway, whereas it was secreted into extracellular environments in V. anguillarum. The results imply that the expression, export and processing mechanism of the protein in E. coli are similar to those in V. anguillarum.     

哈维氏弧菌黑鲷分离株BK-1培养条件优化研究

从患病黑鲷Sparus macrocephalus 的肾脏分离到一致病菌株BK-1,经鉴定为哈维氏弧菌.对哈维氏弧菌BK-1株的最佳生长条件及培养基优化进行了测定.结果表明:不同的培养条件和培养基成份均会影响其产量.哈维氏弧菌最适宜生长条件为:盐度为2%、pH8、温度为30℃;最佳培养基成份为:蛋白胨0.5%,牛肉膏0.75%,甘油0.05%,CuSO40.15 mg/L,CaCl2 0.01 g/L.     

Purification and Inhibition Fungal Growthof Chitinases from Vibrio pacini

0　IntroductionChitinases (EC3.2 .1.14 )areenzymesthathydrolyzechitin ,ahomopolymerofβ 1,4linkedN acetylglucosamine .Thecompletedegradationofchitinchainsisusuallyachievedbyacom plexofenzymesactingtobreaktheinitialchainsintochitooligosac charides .Theseparateenzymesthenacttobreaktheseoligomersdownintotheircomponentmonomers.Thechitinolyticsystemex istsinawiderangeoforganisms,includingbacteria[1 ] ,fungi,higherplants[2 ] ,insects[3] ,crustaceans[4] andsomevertebrates,andplaysvariousrolesbothin…     

海洋鳗弧菌铁载体合成的调控

以一株鳗弧菌pJM1质粒缺陷为研究对象，考察了儿茶酚类铁载体Anguibactin的合成代谢调控。研究发现：在由染色体和质粒共同编码介导的铁载体Anguibactin的合成途径中，其分界点位于2，3－二羟基苯甲酸合成，2，3－二羟基苯甲酸合成之前代谢途径受染色体编码调节,后由质粒编码介导；编码2，3－二羟基苯甲酸的染色体相关基因的表达受环境铁离子浓度的诱导调节。     

对虾病原体鳗弧菌抗血清制备及其血清反应

对鳗弧菌抗血清学制备及其血清反应的研究结果表明，应用免疫注射法，先去除鞭毛抗原，可以获得对虾“黄鳃病”鳗弧菌的高效价的抗血清，其血清效价可以达到５１２０，这为对虾病原菌制备抗血清，提供了一个很好的技术和方法．     

Isolation and characterization of a new species of Vibrio-Vibrio olivaceus sp.nov. from diseased flounder （Paralichthys olivaceus temminck et schlegel）

One kind of pathogenic bacteria was isolated from diseased (or dead) flounder (Paralichthys olivaceus L.) which expressed bacterial septicaemia. The phenotyic information of the 10 pure cultures was studied extensively, including morphological characteristics, colony characteristics in different media, physiological and biochemical characteristics, and the mol%G C ratio of the DNA for representative strain. The results showed that the isolates belonged to a new species of Vibrio. In addition, the representative strains have been re-checked by China Center for Type Culture Collection (CCTCC), the results were the same as ours, the examined strains were also regarded as a new species of Vibrio, and designated as Vibrio olivaceus sp.nov. by its isolated fish (Paralichthys olivaceus) based on its biological properties following Rules of International Code of Nomenclature of Bacteria. In the same time, we conducted studies on the serum homology of isolates, pathogenicity of isolates by experimental infection. The results showed that all the isolates were of serologic similarity and the isolates have strong pathogenicity to flounder. Inspection to pure cultures and isolates from liver of moribund fish from infection experiment by fluorescent antibody technique showed the feasibility of fluorescent antibody technique as an auxiliary method of examination.