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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 kDa.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OmpU和OmpN,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(GenBank 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. 相似文献
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草鱼含信号肽分泌蛋白的预测分析 总被引：3，自引：0，他引：3
采用组合的信号肽分析软件SignalP 3.0,TMHMM 2.0,big-PI,TargetP 1.1和Lipop 1.0对已公布的522个草鱼ORF编码蛋白的N端氨基酸序列进行信号肽分析,同时系统分析了信号肽的类型及结构。结果表明,522个草鱼氨基酸序列中分泌蛋白有61个,占其基因组编码蛋白的11.7%。编码这些蛋白的可读框(ORF)最小值为127 bp,最大值为5032 bp,平均910 bp,引导它们的信号肽长度介于15～32个氨基酸之间,平均为21个氨基酸。通过对草鱼分泌蛋白切割位点信号肽类型分析发现具Ⅰ型信号肽的分泌蛋白有17个,Ⅱ型的有4个。草鱼分泌蛋白中,42个具可预测功能,19个为未知功能蛋白。已知功能的分泌蛋白主要集中于细胞代谢,细胞调控及转运,细胞膜结构,细胞的免疫等领域。 相似文献
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水产动物主要弧菌外膜蛋白结构的比较分析 总被引：1，自引：0，他引：1
用十二烷基肌氨酸钠(Sarkosyl)抽提结合超速离心纯化的方法提取了溶藻弧菌SR1、鱼肠道弧菌HQ010223-1和鳗弧菌S010610-1共3株水产动物致病弧菌及其他11株水产动物主要弧菌和4株非弧菌属细菌的外膜蛋白(Outer membrane proteins,OMPs),通过SDS-PAGE分析其外膜蛋白的组成结构。结果表明,3株致病弧菌分别有12、6和6条外膜蛋白带,且分子量主要集中在32~48kD和66~106kD之间。同时,分析其他11株水产动物主要弧菌和4株非弧菌属细菌共15株细菌外膜蛋白的SDS-PAGE图谱发现,11株弧菌的外膜蛋白图谱比较相似,而与非弧菌属细菌爱德华氏菌、荧光假单胞菌和大肠杆菌则差别明显,且36kD的外膜蛋白为这11株弧菌所共有,而4株非弧菌属细菌的SDS-PAGE图谱则没有36kD的蛋白条带出现。本试验发现,36kD的外膜蛋白存在于所供试的14株弧菌中,而非弧菌属细菌则没有,说明该蛋白有可能是弧菌特异性的外膜蛋白,可作为弧菌属的标志,对于弧菌鉴定有一定的参考价值。 相似文献
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采用十二烷基肌氨酸钠(Sarkosyl)和苯甲基磺酰氟(PMSF)2种方法提取秦皇岛弧菌HQ010712-1(Vibrio qinhuangdaora sp.nov.)外膜蛋白,结果显示 Sarkosyl法提取效果较好,且所提取的主要外膜蛋白分子量为102kD、45 kD、39 kD、36 kD、30 kD、28 kD、24 kD、22 kD;为比较该菌株与弧菌属其他细菌外膜蛋白组分及抗原性异同,以鳗弧菌(Vibrio anguillarum)、副溶血弧菌(Vibrio parahaemolyticus)、溶藻胶弧菌(Vibrio alginolyticus)为对照,电泳图谱显示4种弧菌外膜蛋白的分子量主要集中在22～48 kD之间;利用抗秦皇岛弧菌HQ010712-1血清的免疫印迹表明菌株HQ010712-1外膜蛋白中分子量为45 kD、36 kD的蛋白条带呈现阳性反应,其他3种弧菌外膜蛋白中均有与该抗血清反应的条带,且分子量为36 kD的反应带为菌株HQ010712-1、副溶血弧菌、溶藻胶弧菌共有.本研究旨在为进一步筛选和研究致病性弧菌的共同保护性抗原提供参考. 相似文献
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从哈维氏弧菌(Vibrio harveyi)、溶藻弧菌(V.alginolyticus)、副溶血弧菌(V.parahaemolyticus)克隆、测定了共19株海水鱼类致病性弧菌外膜蛋白OmpK基因序列,探讨其作为海水鱼类致病性弧菌共同抗原的分子基础.根据已知的弧菌外膜蛋白OmpK序列设计1对简并引物,利用聚合酶链式反应(PCR)方法从19株弧菌总DNA中分别扩增得到约800bp外膜蛋白OmpK的基因片段,将其克隆到pDM18-Tvector载体筛选阳性重组子进行序列测定.结果显示,OmpK基因分别含有786bp～849 bp的开放读码框,编码261～282个氨基酸,其核苷酸序列之间的相似性在72%～100%,推测氨基酸序列的相似性为71%～100%,且种内OmpK氨基酸序列的相似性比种间高.序列分析还表明,每一种弧菌OmpK基因都有一段特异性序列,可用于设计核酸探针或特异性引物来诊断、检测哈维氏弧菌等海水鱼致病性弧菌.本研究不仅从基因水平上证实了外膜蛋白OmpK广泛存在于海水鱼致病性弧菌中,而且证明了它们之间具有较高的相似性.由结果推测外膜蛋白OmpK是哈维氏弧菌、溶藻弧菌、副溶血弧菌等致病性弧菌的一种共同抗原,是较好的亚单位疫苗候选成分,为进一步研制广谱的海水鱼类致病性弧菌外膜蛋白基因工程亚单位疫苗提供了理论基础. 相似文献
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分泌蛋白是在细胞内合成后分泌到细胞外起作用的蛋白质,其在许多原生动物寄生虫操纵宿主细胞和影响虫体的毒力中起着重要作用。虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)是一种可感染多种经济虾类的专性细胞内寄生虫,是近年来影响全球对虾养殖生产较严重的病害之一。本研究利用真核生物分泌蛋白预测流程EuSecPred2.0对EHP全基因组的分泌蛋白进行预测,并对分泌蛋白长度、信号肽长度、切割位点处氨基酸分布等信息进行分析,对分泌蛋白功能进行分析。结果显示,分泌蛋白氨基酸长度主要集中在30~400 aa之间;信号肽长度集中在9~32 aa之间;信号肽切割位点处以疏水性氨基酸为主。对信号肽进行基序分析,发现存在基序NV[VT][IK]CA[ED][SA]。对所获蛋白质进行功能注释,发现了多种与微孢子虫黏附侵染、调节细胞周期和免疫反应等相关的关键蛋白。研究结果有助于了解EHP对宿主的侵染机制,同时为进一步明确EHP的致病相关因子提供理论依据。 相似文献
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大菱鲆致病性溶藻弧菌SR1的外膜蛋白及其抗原性分析 总被引：3，自引：0，他引：3
用十二烷基肌氨酸钠(Sarkosyl)抽提结合超速离心的方法提取了一株大菱鲆致病性溶藻弧菌(Vibrio alginolyticus)SR1和其他7株弧菌的外膜蛋白。通过SDS-PAGE图谱分析比较了这8株弧菌外膜蛋白的组成,结果表明,8株弧菌的外膜蛋白电泳一般可得到6-12条条带,其分子量多集中在65-106 kD和28-48 kD,其中36 kD的蛋白带为8株弧菌所共有。用兔抗SR1全菌血清进行Western-blot印迹显示,菌株SR1的外膜蛋白条带中有6条发生了阳性反应,其分子量分别为73 kD、48 kD4、5 kD3、9 kD、36 kD和32 kD。而其他7株弧菌的外膜蛋白与兔抗SR1血清也发生程度不等的阳性反应,这些阳性反应条带的分子量集中在65-73 kD、45-48 kD和36-41 kD之间,其中36 kD的外膜蛋白在8株弧菌中均出现明显的阳性反应,说明36 kD的外膜蛋白是这8株弧菌共有的特异性抗原。 相似文献
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本研究检测了分离自发病大菱鲆、半滑舌鳎及鲤鱼的22株病原鳗弧菌(Vibrio anguillarum)毒力相关基因的携带情况,并建立了病原鳗弧菌的分子生物学检测方法。以PCR方法检测8个毒力相关基因的分布,结果显示,22株病原鳗弧菌均可扩增出6个基因(empA、vah1、vah4、flaA、rtxA和tonB)目的条带,未扩增出virA和angM基因;针对vah4和rtxA设计引物进行双重PCR扩增,同一PCR反应体系可扩增出两条目的条带,灵敏度为2.4×103 CFU/ml,对照菌无任何扩增条带;以vah4设计引物进行LAMP扩增,病原鳗弧菌可扩增出阶梯状条带,呈现阳性反应,6株对照菌无阶梯状扩增条带且呈现阴性反应,LAMP扩增灵敏度为2.4×101 CFU/ml。LAMP检测灵敏度是双重PCR的100倍,LAMP技术与PCR比较,操作简便、快速、灵敏度高且不需昂贵仪器,LAMP检测鳗弧菌的方法更适合于养殖生产实际应用。 相似文献
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利用Western-blot技术,对鳗弧菌L-18株的OMPs和IROMPs的免疫反应性进行了分析,并通过免疫牙鲆比较二者免疫保护力的差异,以期从IROMPs方向探索开发鳗弧菌新型鱼用疫苗,并为研究其免疫原理提供重要的科学依据。结果表明,培养基中加入不同浓度的联铁剂,鳗弧菌L-18株的生长和铁载体的表达出现规律性变化,其中加入100~130μmol·L-1 2,2’-联吡啶为最适合限铁条件,此时铁载体的表达量最高且生长受抑制程度较小。在此条件下,菌株表达出74.3ku和77.4ku的IROMPs,其中77.4ku蛋白具有免疫反应性,为重要抗原。用全菌灭活疫苗、OMPs、IROMPs分别腹腔注射免疫牙鲆7周后攻毒, IROMPs疫苗的相对免疫保护力达到75.9%,远高于OMPs疫苗的51.8%。相对于OMPs,IROMPs的抗体效价显著提高,接近全菌疫苗组的水平,而血清杀菌活力没有显著变化。
关键词：鳗弧菌;铁调节外膜蛋白;牙鲆 相似文献
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摘要：对已分离的1株致病性鳗弧菌W1外膜蛋白图谱进行SDS-PAGE分析，并与8株不同血清型的鳗弧菌外膜蛋白进行比较。结果表明，鳗弧菌W1主要外膜蛋白分别为24kD，38kD，42kD和47kD，主要外膜蛋白图谱与鳗弧菌O1血清型标准菌株VIB1相似。抗生素药敏试验表明该菌对氨苄青霉素、强力霉素、磺胺嘧啶等14种常用药物产生了抗性，只对新生霉素、呋喃妥因、利福平、新霉素等9种药物敏感。研究不同浓度的氨苄青霉素、强力霉素、磺胺嘧啶和庆大霉素对鳗弧菌外膜蛋白表达的影响。结果表明，氨苄青霉素、强力霉素和磺胺嘧啶明显地抑制鳗弧菌42kD的主要外膜蛋白的表达，随着抗菌素浓度的增加，该外膜蛋白的表达量逐渐减少甚至消失，而庆大霉素浓度的变化对其表达没有明显影响。对该42kD主要外膜蛋白进行N末端分析表明，其N末端序列为EAPTAINS，与已发表的细菌其他外膜蛋白序列没有同源性。 相似文献
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Vibrio anguillarum is an important bacterial fish pathogen responsible for epizootics in both marine and freshwater fish worldwide. Studies on pathogenic V. anguillarum has shown that its virulence is mediated by a 65 kb endogenous pJM1-like plasmid, which encodes an efficient iron uptake system. The plasmid-free derivative of wild V. anguillarum was found to be greatly attenuated and elicited a good protection against wild V. anguillarum in fish. In this study, a plasmid-free derivative MVAV6201, an effective live vaccine candidate, was used as a carrier strain to achieve the secretory delivery of recombinant proteins in V. anguillarum. The secretion mechanism was based on the Escherichia coli alpha-haemolysin (HlyA) transport system. The recombinant proteins were fused with the alpha-haemolysin secretion signal (HlyAs) and expressed from the commonly used HlyA secretion vector pMOhly1. Two HlyAs-tagged recombinant proteins, GFP-HlyAs and AngE-HlyAs, were constructed and their secretion characters in V. anguillarum investigated. In the case of GFP-HlyAs, nearly 70% of the total fusion protein was efficiently secreted into culture supernatant, and in the case of AngE-HlyAs, the secretion efficiency was determined to be about 300 microg L(-1) by Western blotting. 相似文献
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Abstract. Specificities of polyclonal salmon antisera made against the fish pathogens Vibrio salmonicida and Vibrio anguillarum were studied. Using ELISA and Western blot techniques, antisera made against V. salmonicida or V. anguillarum serovar 1 demonstrated high responses against the homologous bacterium or its isolated LPS. In contrast, antisera obtained after immunization with V. anguillarum serovar 2 displayed low antibody titres against homologous antigens. Elcctrophoretic transfer of SDS-PAGE separated V. salmonicida LPS antigen to nitrocellulose strips and subsequent immunostaining with salmon antisera revealed a strong reaction exclusively in the low molecular weight region (<14kD). On the other hand, immunoblots of V. anguillarum LPS preparations using salmon immunesera raised against this species showed a heterogenous staining pattern ranging from high to medium LPS-size. In addition, most of the salmon antisera made against V. anguillarum serovar 2 also reacted with a low molecular weight LPS antigen band. 相似文献
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Electrophoretic and immunochemical analysis of surface antigens of the fish pathogens Vibrio salmonicida and Vibrio anguillarum 总被引：3，自引：0，他引：3
J. BØGWALD K. STENSVÅG J. HOFFMAN S. ESPELID K. O. HOLM T. JØRGENSEN 《Journal of fish diseases》1990,13(4):293-301
Abstract. Outer membranes and lipopolysaccharides of the marine fish pathogens Vibrio salmonicida and Vibrio anguillarum were isolated. SDS-PAGE profiles of purified LPS preparations from V. salmonicida revealed a broad low molecular weight band, whereas V. anguillarum LPS profiles demonstrated both a low-molecular band and several weaker high-molecular weight bands. Hydrolysis of V, salmonicida and V. anguillarum LPS separating the polysaccharide chain from the lipid A part and subsequent gel-chromatography suggests a polysaccharide molecular weight of ca. 1000 ('rough type' LPS) and ca. 6000 ('smooth type' LPS), respectively. Western blot of V. salmonicida outer membrane preparations and purified lipopolysaccharides and subsequent immunostaining with mouse monoclonal antibodies was performed. Eleven out of 15 monoclonal antibodies made against V. salmonicida cells reacted with one broad antigen-band in the low molecular weight region of both outer membrane and LPS profiles, corresponding to the LPS region. The previously reported outer surface antigen, VS-P1 from V. salmonicida , was observed to carry LPS epitopes as revealed by binding of monoclonal antibodies to VS-P1 as well as purified LPS preparations. These results strongly suggest that the VS-P1 antigen is a complex of both protein and LPS molecules. 相似文献
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Abstract. Sixty-eight strains of Vibrio anguillarum , five of V. ordalii and the type strains of V. alginolyticus, V. carchariae, V. damsela and V. parahaemolyticus were compared using the API 20E gallery. Within the V. anguillarum strains, distinct groups could be separated mainly on the basis of their reaction on indole production and the fermentation of amygdalin and arabinose. Vibrio ordalii , the former V. anguillarum biotype 2, could easily be separated from V. anguillarum and from the other fish pathogenic Vibrio spp. 相似文献
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采用试管二倍稀释法测定二氟沙星对鳗弧菌W-1、副溶血弧菌1614和溶藻弧菌1833的最小抑菌浓度(MIC);采用菌落计数法测定二氟沙星对鳗弧菌、副溶血弧菌和溶藻弧菌的抗菌后效应(PAE)。结果显示,二氟沙星在1×MIC、2×MIC和4×MIC浓度时,对鳗弧菌W-1、副溶血弧菌1614和溶藻弧菌1833的PAE分别为:0·64、1·09和2·16h;0·74、1·73和2·64;0·54、1·08和2·05h。二氟沙星对这3种弧菌具有明显的抗菌后效应,并且随着二氟沙星浓度的增加,抗菌后效应的时间也明显延长。 相似文献
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Live, attenuated Vibrio anguillarum strains can serve as vectors for the delivery of heterologous antigens for development of multivalent recombinant vaccines. Based on the outer membrane anchoring elements of V. anguillarum , we have previously constructed several efficient surface display systems Lpp-Omporf1, Lpp-OmpU, Lpp-Omp26La, Wza-Omporf1, Wza-OmpU and Wza-Omp26La. In this study, with these constructed surface display systems, a putative antigen protein EseB from pathogenic Edwardsiella tarda was successfully expressed on the surface of an attenuated V. anguillarum strain to get multivalent vaccine candidates. Further immune protection evaluation in zebra fish ( Danio rerio ) demonstrated that the V. anguillarum EseB-display strain AV/pW-26La-B could trigger full protection against V. anguillarum infection and early protection against E. tarda infection in the immunized fish. These results suggest that surface display of heterologous protective antigens in attenuated V. anguillarum could be used as a tool to develop potential V. anguillarum vector vaccine. 相似文献
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Abstract. Vibrio anguillarum isolates were sampled from cod fry showing clinical signs of vibriosis at different cod farms along the western coast of Norway. Except for one isolate, all were shown to cause mortality in laboratory challenge experiments. Using biochemical analysis and specific rabbit antisera (O1–O5), all the pathogenic isolates were classified as typical V. anguillarum serotype O2. SDS-PAGE and Western blot analysis of both whole bacterial cells and purified lipopolysaccharides (LPS) showed that the LPS structures were heterogeneous. ELISA analyses using adsorbed antisera identified the presence of two different subgroups within serogroup O2, which correspond with O2a/O2α and O2b/O2β described previously. 相似文献