鳗弧菌、溶藻胶弧菌外膜蛋白的分离及特性

分别用SDS、Sarkosyl及PMSF法分离制备鳗弧菌（Vibrio anguillarum）、溶藻胶弧菌（Vibrio alginolyticus）主要外膜蛋白（MOMP），通过SDS-PAGE分析比较2种弧菌主要外膜蛋白的组成结构。结果表明，SDS、Sarkosyl和PMSF法分离提取的鳗弧菌和溶藻胶弧菌外膜蛋白中，SDS-PAGE电泳图谱不同，鳗弧菌外膜蛋白分子量为27-138kD；溶藻胶弧菌外膜蛋白分子量为27-104kD，其中，45kD、30kD和27kD外膜蛋白为鳗弧菌和溶藻胶弧菌所共有。经比较，Sarkosyl法对2种弧菌外膜蛋白的提取效果较好。对3种方法提取的2种弧菌的主要外膜蛋白进行SDS PAGE后，分别与吸附后的兔抗鳗弧菌、抗溶藻胶弧菌血清的Western-blotting印迹显示，兔抗鳗弧菌血清与其菌株的外膜蛋白主要有3条免疫反应带，其分子量分别为97kD、51kD和30kD，说明其可能与鳗弧菌抗原的特异性有关；兔抗溶藻胶弧菌血清与其菌株的外膜蛋白主要有2条免疫反应带，其分子量分别为51kD和27kD，说明可能与溶藻胶弧菌抗原的特异性有关，而51kD外膜蛋白可能是2种弧菌共有的特异性抗原。     

水产动物主要弧菌外膜蛋白结构的比较分析

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。     

牙鲆秦皇岛弧菌HQ010712-1外膜蛋白及其抗原性分析

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.     

大菱鲆致病性溶藻弧菌SR1的外膜蛋白及其抗原性分析

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。     

创伤弧菌、溶藻弧菌外膜蛋白特性的比较研究

对用Sarkosyl法分离的创伤孤菌、溶藻弧菌、副溶血弧菌、鳗弧菌的外膜蛋白进行了初步的比较分析.这4种弧菌外膜蛋白的SDS-PAGE和Western blotting的图谱有相似性亦有差异.SDS-PAGE显示,4种弧菌中除副溶血弧菌外均能分离到47、38 ku的外膜蛋白;创伤弧菌和溶藻弧菌存在4种共同的外膜蛋白,大...     

3株水产动物病原弧菌主要分泌性蛋白的鉴定及其分泌性序列的分析

从3株水产动物病原菌鳗弧菌MN、鳗弧菌3101、费氏弧菌培养液中分别获取了分泌性蛋白,进行SDS-PAGE,对表达量较高的5条蛋白区带进行MALDI-TOF/TOF质谱鉴定表明,它们分别是鳗弧菌MN金属蛋白酶(empA-MN)、鳗弧菌3101金属蛋白酶(empA-3101)、锌金属蛋白酶(zincempA)、费氏弧菌VFMJ11_1094蛋白(VFMJ11_1094)和费氏弧菌ES114的外膜蛋白(OMP).根据所鉴定的序列设计引物,分别从鳗弧菌MN、鳗弧菌3101和费氏弧菌中扩增出相应基因,克隆后测定emp-MN、empA-3101、zinc empA、VFMJ11-1094和OMP相应基因的序列,经在线软件SignaIP 3.0分析,确定emp-MN、empA-3101、zinc empA、VFMJ11-1094和OMP均存在不同的分泌性信号肽序列angMN-35、ang3101-35、ang3101-25、vf-38和vf-23,通过在线软件PSORT分析表明,所有信号肽均定位在细胞的周质空间或细胞外膜上.     

鱼肠道弧菌外膜蛋白抗原性分析

采用十二烷基肌氨酸钠法和苯甲基磺酰氟法提取鱼肠道弧菌外膜蛋白.SDS-PAGE图谱显示,PMSF提取的鱼肠道弧菌有19条带,Sarkosyl法提取的鱼肠道弧菌有14条带,其中111、105、87、78、61、58、53、48、45、40、36、33、32、31 kD蛋白带为2种方法共同条带,101、55、42、27、25 kD为PMSF法特有条带.此外,以制备的兔抗鱼肠道弧菌全菌血清为第一抗体,应用Western-blotting技术分析了鱼肠道弧菌外膜蛋白的抗原性,结果显示分子量为106、87、61、58、55、42、36、32 kD的8条蛋白带发生了免疫反应.     

对越冬中国对虾烂眼病的初步研究

本文对越冬中国对虾烂眼病的病因,症状,药物治疗,分离菌9013对抗菌药物敏感性做了初步研究,结果表明:烂眼病的发生初步断定是由革兰氏阴性,弧状,端生单根鞭毛的孤菌9013感染所致。大蒜、痢特灵、漂白粉对烂眼病有不同程度的治疗作用。分离苗9013对新诺明、红霉素、白霉素、吡哌酸、土霉素、先锋霉素、氯霉素、四环素显示高度敏感,对丁氨卡那霉素、氨苄青霉素、庆大霉素、新霉素、氯唑青霉素、链霉素、头胞菌素、痢物灵、卡那霉素显示中度敏感,对磺胺嘧啶、青霉素,新青Ⅱ号、羧苄青霉素、磺苄霉素显示耐药性。     

哈维氏弧菌外膜蛋白OmpK基因的克隆及原核表达

根据哈维氏弧菌外膜蛋白OmpK的基因序列设计一对引物，应用聚合酶链式反应（PCR）方法，从分离自患病大黄鱼的哈维氏弧菌基因组中扩增获得一段约800bp的序列。将其克隆到pGEM-Teasy载体，测序结果证明该序列是哈维氏弧菌外膜蛋白OmpK基因。用PCR方法去除其信号肽序列，定向克隆到原核表达载体pGEX-4T-2构建重组表达质粒pGEX-4T-OmpK。IPTG诱导后能够在大肠杆菌BL21中高效表达分子量约为53kD的GST-OmpK融合蛋白。用纯化后的融合蛋白免疫新西兰兔获得了高效价的抗血清。Western-blotting分析表明，它与从哈维氏弧菌中提取的约27kD的外膜蛋白能够发生特异反应，提示外膜蛋白OmpK可能是哈维氏弧菌的重要保护性抗原之一。     

鱼肠道弧菌外膜蛋白疫苗对牙鲆免疫效果的初步研究

为研究鱼肠道弧菌外膜蛋白疫苗对牙鲆的免疫效果,用该种疫苗对牙鲆进行了免疫试验.给牙鲆注射鱼肠道弧菌外膜蛋白疫苗,饲养25 d后分别用鱼肠道弧菌、鳗弧菌和溶藻弧菌进行攻毒,20 d后分别统计各组鱼的免疫保护率.结果表明,该疫苗对鱼肠道弧菌、鳗弧菌和溶藻弧菌的免疫保护率分别为84.6%、30.8%、33.3%.     

Cellular components of probiotics control Yersinia ruckeri infection in rainbow trout, Oncorhynchus mykiss (Walbaum)

Subcellular components of the probiotics Aeromonas sobria GC2 and Bacillus subtilis JB-1, when administered to rainbow trout, Oncorhynchus mykiss , conferred protection against a new biogroup of Yersinia ruckeri . Thus, intraperitoneal or intramuscular injection of rainbow trout with cell wall proteins (CWPs), outer membrane proteins (OMPs), lipopolysaccharides (LPS), whole cell proteins (WCPs) and live cells followed by challenge on day 8 with Y. ruckeri led to 80–100% survival compared with 10% survival in the controls. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of WCPs and OMPs from GC2 had 10 and 5 variable protein bands in comparison to 11 and 5 bands in the WCPs and CWPs from JB-1. Proteomic analyses were employed following SDS-PAGE to categorize one dominant protein of 104.7 kDa from the CWPs of JB-1 and equated it with ' Bacillus spp. endoglucanase' with a Mascot score >69. These results point to the potential of using cellular components of probiotics for protection of fish against bacterial diseases.     

Electrophoretic and immunochemical analysis of surface antigens of the fish pathogens Vibrio salmonicida and Vibrio anguillarum

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.     

Proteomic analysis of the sarcosine-insoluble outer membrane fraction of Flavobacterium columnare

Outer membrane proteins (OMPs) of bacteria are key molecules interacting with the host environment. Flavobacterium columnare, a pathogen-causing columnaris disease of fish worldwide, was studied in order to understand the composition of its OMPs. The sarcosine-insoluble membrane fraction of the OMPs was analysed using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in combination with reverse-phase high-performance liquid chromatography-tandem mass spectrometry (RP-HPLC MS/MS). Thirty-six proteins were identified, including proteins involved in cell wall/membrane biogenesis, specific transport of various nutrients and in essential metabolism. The present study is the first report on the OMPs of F. columnare, and may serve as the basis for understanding the pathogenesis of the bacterium.     

淋巴囊肿病毒结构蛋白及其抗原性分析

病鱼为威海水产养殖场感染淋巴囊肿病的牙鲆（Paralichthys olivaceus）,收集病鱼的囊肿组织,匀浆破碎,采用差速离心和蔗糖密度梯度离心方法,分离纯化淋巴囊肿病毒粒子.负染后,电镜观察证实获得的病毒纯度高,杂质极少,病毒粒子呈近似于圆形的多角形,结构完整.纯化的淋巴囊肿病毒粒子经SDS-PAGE,硝酸银染色后,电泳图谱清晰显示病毒结构蛋白带共有22条,且分子量主要集中在123～26 kD.应用Western blotting法分析病毒结构蛋白的抗原性,结果显示,分子量分别为123.55 kD、65.292 kD和54.438 kD的3条蛋白带发生了免疫反应,其中分子量为65.292 kD的蛋白带反应强度明显高于其他2条蛋白带.本研究旨为确定淋巴囊肿病毒主要衣壳蛋白提供基础依据.[中国水产科学,2006,13（3）：415-420]     

Antibody specificities in Atlantic salmon, Salmo salar L., against the fish pathogens Vibrio salmonicida and Vibrio anguillarum

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.