产低温碱性蛋白酶黄海黄杆菌YS-9412-130高产菌株的选育

以黄海黄杆菌YS－9412－130菌株为出发株,经亚硝基胍、硫酸二乙酯、紫外线（UV）与微波（MI）复合诱变和自然选育,获得1株产低温碱性蛋白酶高产稳定的突变株YS－9412－130－SW1－104,其产低温碱性蛋白酶为出发株的16倍。在摇瓶发酵培养条件下,酶活力达2320U／ml(20℃测定）,该诱变株具有Ref^r、Str^r、Amp^r和Met^-、Lys^-标记。突变株与出发菌株在细胞形态上比较,结果表明二者没有显著差异。     

产低温碱性蛋白酶黄海黄杆菌YS-9412-130的复合诱变原生质体选育

以黄海黄杆菌YS－9412－130突变株SW1－104为出发菌,在原生质体形成和再生的最佳条件下制备原生质体,对原生质体进行复合诱变,对大量再生突变株进行筛选和淡水驯化,最终获得了高产、稳定的碱性蛋白酶产生菌SW2－104,在自来水培养基中能够大量产酶,产酶活力为3910U／ml。从而解决了黄海黄杆菌YS－9412－130低温碱性蛋白酶大规模工业化生产设备和产业化区域的局限性。     

黄海黄杆菌YS-9412-130低温碱性蛋白酶的制备工艺研究

对黄海黄杆菌所产低温碱性蛋白酶的工业生产技术和电泳纯级试剂酶的分离纯化工艺进行了实验。按照设计的中试生产技术，每吨发酵液可得酶粉20kg，粗酶比活大于20×10     

黄海黄杆菌YS-9412-130低温碱性蛋白酶性质鉴定

对黄海黄杆菌YS-9412-130菌株产低温碱性蛋白酶的理化性质研究表明，该酶由256个氨基酸组成，分子量为33000Dar，等电点pI为9.45，米氏常数Km为5×10-3mmol/L；酶的最适作用pH范围为9.5～10.5，最适作用温度30℃，具有一定的抗氧化稳定性。Ca2+、Mn2对酶有激活作用，而Hg2+、Ag+对酶有抑制作用。DFP、NBS严重抑制酶的活性,而同时该酶也能被EDTA抑制。结果表明该低温碱性蛋白酶为一新型的丝氨酸蛋白酶。     

黄海黄杆菌YS-9412-130低温碱性蛋白酶发酵过程动力学研究

应用自动控制发酵设备，对黄海黄杆菌YS－9412－130碱性蛋白酶发酵动力学和工艺条件进行了研究。从基本的动力学概念和方程出发，通过分批发酵试验摸索了黄海黄杆菌YS－9412－130生长与代谢的基本规律，证明了发酵过程中低温碱性蛋白酶的形成为生长部分关联型。采用补料分批培养方法限制生长基质浓度，确定其一系列数值，从而推导出细胞生长与产物合成的动力学数学模型。     

黄海黄杆菌YS-9412-130低温碱性蛋白酶多克隆抗体的制备

利用纯化的黄海黄杆菌海洋低温碱性蛋白酶（Marine Low-temperature Alkaline Protease,MLAP),采用背皮内多点注射和皮下组织注射免疫相结合的方法对新西兰白兔进行免疫,制备了多克隆抗体,并对多克隆抗体进行了纯化。用ELISA的方法对其效价检测表明,其效价为128000。作者所做的Western印迹实验证明了抗原抗体的结合具有高度的特异性。     

黄海黄杆菌YS-9412-130产酶发酵条件的优化

以产低温碱性蛋白酶的黄海黄杆菌YS－9412－130突变株SW2－104为培养菌株,进行碳源、氮源、无机盐、起始pH值、培养时间、接种量和接种龄等发酵条件的优化实验。实验结果证明,在以1％葡萄糖为碳源,以3.5％豆饼粉为氮源,无机盐：0.4%Na2HPO4、0.03%KH2PO4、0.02%MgSO4、0.1%Na2CO3、0.2?Cl2,起始pH值7.0,接种12h种龄的种子4％,20℃、250r/min的条件下在旋转摇床中培养36h,菌株产酶活性最高。     

柱状黄杆菌胶原蛋白酶的克隆与表达

利用PCR方法从柱状黄杆菌(Flavobacterium.columnare)G4株中克隆了1个胶原蛋白酶基因(GenBank登录号EF501979).同源性比对发现该基因与柱状黄杆菌的另外1种胶原酶基因(GenBank登录号EAZ95511.1)最为相近,有84%的一致性和93%的相似性.该属的另外2种细菌,约氏黄杆菌(Flavobacterium.johnsoniae)和嗜冷黄杆菌(F.psychrophilum)都有与该胶原酶基因相似的基因,其相似性分别为92%和83%.该基因经KpnI和SalI酶切后连接到表达载体pET-32a上,转人大肠杆菌BL21(DE3)plysS内进行表达,经SDS-PAGE电泳后显示重组融合蛋白在44 kD处有明显表达带,与预期分子量大小一致,且主要以不溶的包涵体形式存在,变性条件下利用His·Bind树脂成功纯化了融合蛋白,将其免疫家兔,获得兔抗柱状黄杆菌胶原蛋白酶抗体,Western blotting表明该胶原酶在柱状黄杆菌的胞内和胞外都存在.这些结果为进一步研究这种胶原蛋白酶的功能及柱状黄杆菌的致病机理奠定了基础.     

黄海黄杆菌YS-9412-130低温碱性蛋白酶活性必需基团分析

用化学修饰法结合酶的紫外吸收光谱变化研究黄海黄杆菌YS－9412－130分泌的海洋低温蛋白酶的功能基团性质，结果表明，羟基、咪唑基、ε－氨基及胍基均与酶活性有关，而羟基、巯基与酶活性无关。酶的性质与丝氨酸蛋白酶有很大的相似性。     

黄海黄杆菌YS-9412-130低温碱性蛋白酶的应用研究Ⅱ.——洗涤剂用包覆型酶的配伍特性与应用效果评价

论述了洗涤剂中的低温碱性蛋白酶的物理和化学特性、去污性能及它与常规洗涤助剂的配伍性。实验结果表明，该包覆型酶最适反应温度为35℃，pH为10。在温度40℃以下、pH5-11范围内均具有良好的稳定性，同时对低温有突出的适应性和抗氧化稳定性；与常规洗涤剂各成分配伍性良好，且低温条件下能有效降解蛋白污渍，主要性能均优于国内外同类产品。     

Inhibitory activity of Pseudomonas sp. on Flavobacterium psychrophilum, in vitro

A Pseudomonas sp. isolate MSB1 efficiently inhibited the growth of Flavobacterium psychrophilum of different serotypes on agar medium. A significant difference in the inhibition was observed between isolates of the less virulent Fp^T serotype compared to the Fd and Th serotypes. In broth coculture experiments, a low number of cells of MSB1 inhibited and outcompeted the F. psychrophilum cells. Also cell‐free culture supernatant of MSB1 clearly repressed the growth of F. psychrophilum. A chromoazurol S assay suggested that MSB1 produced efficient siderophores, which most probably were responsible for the iron deficiency in the supernatant. The limited growth of F. psychrophilum in the supernatant was found to be partly because of the lack of available iron, but the results also indicated that some other mechanisms were probably involved in the observed inhibition. A potential use of MSB1 as a probiotic in rainbow trout aquaculture, especially in early life stages of the fish, is suggested, but future in vivo experiments needs to be carried out to verify this suggestion. This study also indicates a low iron acquisition efficiency of F. psychrophilum, compared to other examined bacterial fish pathogens.     

Cross-protection of a live-attenuated Flavobacterium psychrophilum immersion vaccine against novel Flavobacterium spp. and Chryseobacterium spp. strains

For salmonid producers, a common threat is Flavobacterium psychrophilum. Recent advancements in bacterial coldwater disease (BCWD) management include the development of a live-attenuated immersion vaccine that cross-protects against an array of F. psychrophilum strains. Emerging family Flavobacteriaceae cases associated with clinical disease have been increasing, including pathogenic isolates of Flavobacterium spp. and Chryseobacterium spp. The cross-protective ability of a live-attenuated F. psychrophilum vaccine was determined against three virulent Flavobacteriaceae isolates. Juvenile rainbow trout were vaccinated, developed high F. psychrophilum-specific antibody titres and were challenged with Chryseobacterium spp. isolates (S25 and T28), a Flavobacterium sp. (S21) isolate, a mixed combination of S21:S25:T28, and a standard virulent F. psychrophilum CSF259-93 strain. Results demonstrated strong protection in the CSF259-93 vaccinated group (relative per cent survival (RPS)=94.44%) when compared to the relevant CSF259-93 controls (p < .001). Protection was also observed for vaccinated fish challenged with the S21:S25:T28 mix (RPS = 85.18%; p < .001). However, protection was not observed with the S21, S25 or T28 isolates alone. Analysis of whole-cell lysates revealed differences in protein banding by SDS-PAGE, but conserved antigenic regions by Western blot in S25 and T28. Results demonstrate that this live-attenuated vaccine provided protection against mixed flavobacterial infection and suggest further benefits against flavobacteriosis.     

Pathology and immunohistochemistry in three species of salmonids after experimental infection with Flavobacterium psychrophilum

Rainbow trout, Oncorhynchus mykiss (Walbaum), sea trout, Salmo trutta L., and Atlantic salmon, Salmo salar L., were experimentally infected with Flavobacterium psychrophilum in order to evaluate any species differences in susceptibility to the bacterium. Furthermore, differences in pathological changes and distribution of the bacteria in internal organs were studied. The bacteria were injected intraperitoneally in two doses, high dose (Hd) 1 x 10(7) colony forming units (CFU) fish(-1) and low dose (Ld) 1 x 10(6) CFU fish(-1). The mortalities in the Ld groups varied between 0 and 7.5% and in the Hd groups between 55-70%. No significant differences in mortality between the species were recorded. Clinical signs and pathological findings were similar in the three species and in accordance with those of rainbow trout fry syndrome. Rainbow trout showed more pronounced lesions in the spleen compared with the other species. Necrosis of renal tubular epithelium and haematopoietic tissue was most prominent in rainbow trout and Atlantic salmon. Intracellular eosinophilic droplets in the kidney tubular epithelium were a prominent finding in rainbow trout and sea trout surviving the infection. The distribution of the bacteria in internal organs was similar in the three species, as studied with immunohistochemistry.     

Antimicrobial susceptibility pattern of Flavobacterium columnare isolates collected worldwide from 17 fish species

Flavobacterium columnare is the causative agent of columnaris disease in diverse fish species worldwide. Although columnaris is an important disease, the antimicrobial susceptibility pattern of F. columnare is not well studied. Thus, the purpose of this study was to test the in vitro antimicrobial susceptibility of 97 F. columnare isolates collected worldwide between 1987 and 2011 from 17 fish species. The broth microdilution technique was utilized for reliable testing of these fastidious organisms. None of the isolates displayed acquired resistance to florfenicol, gentamicin, ormetoprim‐sulfadimethoxine and trimethoprim‐sulfamethoxazole. Acquired resistance to chloramphenicol was detected in 1%, to nitrofuran in 5%, to oxytetracycline in 11% and to enrofloxacin, flumequine and oxolinic acid in 10%, 16% and 16% of the isolates, respectively, as reflected by a bimodal or trimodal distribution of their minimum inhibitory concentrations (MICs). One isolate showed acquired resistance towards several antimicrobial agents including erythromycin. Another isolate revealed acquired resistance towards – amongst others – ampicillin. The isolates displaying acquired resistance originated from ornamental fish species or Vietnamese catfish, except for two isolates coming from wild channel catfish in which acquired resistance was encountered towards oxytetracycline only. Fifty per cent of the resistant isolates from ornamental fish were shown to have acquired resistance against three classes of antimicrobial agents, assigning these isolates as multiple resistant. These data might indicate less prudent use of antimicrobials especially in ornamental fish species.     

The influence of salmon surface mucus on the growth of Flavobacterium columnare

Flavobacterium columnare is the causative agent of columnaris disease. The presence of lesions on the gills, skin and fins of diseased fish suggests that F. columnare is able to utilize fish skin mucus as a substrate for growth and that exposure to this material would alter the expression of genes involved in the colonization of the outer surfaces of the fish. Growth, biofilm formation, extracellular protease production and changes in protein expression of F. columnare strain C#2 cultured in media supplemented with juvenile Atlantic salmon skin mucus were compared with the same media without mucus. C#2 was able to grow by using mucus as the sole nutrient source. Growth in mucus-containing media induced cells to grow as a biofilm and extracellular protease activity increased in mucus-containing cultures. SDS-PAGE protein profiles showed that expression of six extracellular proteins increased in mucus-containing media. These results demonstrate that salmon surface mucus promotes the growth of F. columnare and that exposure to mucus alters the growth characteristics of this bacterium with regard to protease production and biofilm formation. Further characterization of mucus-induced physiological changes will increase our understanding of the basis of virulence of this economically important fish pathogen.     

中国裸身虾虎鱼属(Gymnogobius)一新种(鲈形目：虾虎鱼科)

以头部感觉管孔及颊部感觉乳突的排列规律为主要分类依据,综合形态特征,研究了采自浙江省舟山岛河溪的裸身虾虎鱼属(Gymnogobius)的一新种：舟山裸身虾虎鱼Gymnogobius zhoushanensis sp. nov. 。新种的形态特征及性状与产于浙江省苍南县的横带裸身虾虎鱼 Gymnogobius transversefasciatus (Wu & Zhou, 1990)十分相似,头部均具4个感觉管孔 (C,D,F,G),颊部具1条眼下感觉乳突线(L2),3条水平状的纵行感觉乳突线(L3, L4, L5)。但该新种具有口小,上颌骨后端仅伸达眼前缘的下方;纵列鳞、横列鳞和背鳍前鳞较少;头部密具许多小黑点的特征。 关键词： 虾虎鱼科;裸身虾虎鱼属;新种;舟山     

柱状黄杆菌乙酰辅酶A合成酶基因及其上游调控序列分析

柱状黄杆菌(Flavobacterium columnare)是世界范围内危害淡水鱼类的柱形病的病原。目前对该病原菌遗传操作系统的研究进展较慢,而寻找高效稳定的启动子来调控外源基因在细菌体内的表达,有可能促进该细菌遗传操作系统的构建。本研究获得了柱状黄杆菌乙酰辅酶A合成酶基因(acetyl-coenzyme A synthetase gene,acs)的编码序列及其上游调控序列,该基因全长2 323 bp,编码635个氨基酸。通过序列分析,发现在该基因起始密码子ATG的上游存在核糖体结合位点(ribosome biding site,RBS)序列TAAAA,和启动子–7和–33的保守基序TATTTTCG和TTG。将acs的上游调控序列(promoter sequence,Pacs)置于氯霉素抗性基因(chloramphenicol acetyltransferase,cat)的上游并导入柱状黄杆菌G4株后,cat基因得以表达,并使宿主细胞产生稳定的氯霉素抗性。通过5′RACE技术,确定了外源的cat基因和内源的acs基因的转录起始位点都是位于起始密码子上游46 bp处的T。通过删减分析调控序列Pacs,发现起...