草鱼肠道拮抗性芽孢杆菌的冻干保护剂优化研究

自草鱼肠道内提取的病原拮抗性芽孢杆菌能有效提高草鱼免疫能力,从而促进草鱼的健康生长。为提高本试验病原拮抗性芽孢杆菌保存活力,采用L_9(3~4)正交试验法,以病原拮抗性芽孢杆菌冻干保护剂在冷冻干燥后菌体成活率为考察指标,对麦芽糊精、甘氨酸和海藻糖3个变量因素进行考察。结果表明,当以冻干后菌体成活率为考察指标时,各因素对菌体冻干粉的保存活性作用影响为麦芽糊精甘氨酸海藻糖,3种赋形剂对菌体保护最佳配方为A_1B_1C_3,即2%麦芽糊精,0.5%甘氨酸,6%海藻糖。通过该试验方案得到了病原拮抗性芽孢杆菌冻干保护剂的最优配方。     

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%.     

低场核磁共振技术在鱼用冻干疫苗保护剂筛选中的应用

为筛选适合鱼类冻干疫苗中使用的保护剂,采用低场核磁共振技术(LF-NMR)对冻干后不同保护剂中的水分分布和迁移规律进行了分析。根据水中氢质子的弛豫时间,可将保护剂中的残留水分为结合水、不易移动水和自由水3种形态。结果显示:冷冻干燥过程中二次干燥温度对甘露醇和蔗糖中残留水的分布有较大影响,随着温度从4℃升高到37℃,大部分自由水解吸而小部分转化为结合水。当冻干后的样品暴露在相对湿度43%和温度25℃的环境中时,大部分保护剂吸收水分,并将其固定为结合水,而甘露醇中的部分结合水则转变为自由水,并解吸到环境中。不同冻干保护剂中的水分分布和迁移并不相同,代表其具有不同的水分结合能力。该研究结果有助于鱼用冻干疫苗中保护剂的筛选和冻干工艺的优化,以便获得更加稳定的疫苗产品。     

海洋侧孢短芽孢杆菌S-12-86溶菌酶的分离纯化与冻干技术研究

海洋微生物溶菌酶发酵液经低温离心、超滤浓缩、乙醇提取、Superdex 75 10/300凝胶层析和反相高效液相色谱层析纯化得到电泳纯海洋溶菌酶,分子量为17.1 kD,比活达到3 987.7 U/mg,纯度提高41.98倍,活力回收率为21.7%。对该酶冷冻干燥过程的研究结果表明,海藻糖、蔗糖和麦芽糖对该酶均有一定的冻干保护作用。其中,以海藻糖的保护效果最佳。0.5 mol/L海藻糖与20 mg/ml吐温80复合作为冻干保护剂,使冻干后的溶菌酶活性维持在95%以上,为该酶进一步研究和应用提供了稳定的酶制剂。     

鳗弧菌和溶藻弧菌二联疫苗对大菱鲆的免疫效果

将鳗弧菌（Vibrio anguillarum）和溶藻弧菌（V.alginolyticus）分别用0.3%福尔马林灭活,制备成鳗弧菌和溶藻弧菌2种单苗,并将2种菌等量配制成二联疫苗,单次腹腔注射免疫大菱鲆（Scophtalmus maximus）.通过检测免疫后替代途径补体活力、溶菌酶活力、抗体凝集效价的变化并分别进行攻毒实验比较疫苗的免疫效果.结果显示,3种疫苗对所测定的各免疫指标都有一定的促进作用,但二联疫苗后期效果低于2种单苗.二联疫苗对鳗弧菌和溶藻弧菌的免疫保护率分别为83.52%和83.24%,而鳗弧菌单苗对鳗弧菌攻毒、溶藻弧菌单苗对溶藻弧菌攻毒的免疫保护率分别为80.37%和74.89%.可以认为2株菌具有制备二联疫苗的可能性.[中国水产科学,2006,13（3）：397-402]     

多效价载体疫苗免疫大菱鲆效果评价

采用注射与浸泡2种方法,对构建的多效价载体疫苗MVAV6203A-1进行了免疫保护效果和血清效价的研究.结果显示,用多效价载体疫苗注射免疫大菱鲆(Scophthalmus maximus),可同时获得对鳗弧菌(Vibrio anguillarum)和嗜水气单胞菌(Aeromonas hydrophila)的免疫保护效果,免疫相对保护率分别为80.6％和77.0％；用ELISA方法测定血清效价的结果显示,免疫MVAV6203A-1后大菱鲆血清效价最高达到2 048,均值为891,明显高于对照组的97；用多效价载体疫苗浸泡免疫大菱鲆后,对鳗弧菌与嗜水气单胞菌的免疫相对保护率分别为62.2％和11.7％.血清效价分析显示,实验组与对照组的均值均为64.上述结果表明,所构建的疫苗MVAV6203A-1可有效提高大菱鲆对鳗弧菌和嗜水气单胞菌的免疫力,并且免疫和攻毒的途径可能是影响免疫保护效果的因子.本研究旨在为鳗弧菌与嗜水气单胞菌引起的鱼类疾病防治提供技术支撑.     

大黄鱼仔、稚、幼鱼发育阶段的脂肪酸组成及其变化

为了探讨真空冷冻干燥过程参数对冻干时间和能耗的影响，以墨西哥湾扇贝为研究对象，设计二次正交回归组合试验方案，采用SAS统计软件对试验数据进行回归分析，建立了真空冷冻干燥时间和能耗的二次多元回归模型，并对二次多元回归模型进行了F检验；利用降维法定性地分析了物料厚度、干燥室压强和加热板温度3个主要过程参数对真空冷冻干燥时间和能耗的影响。结果表明，物料厚度、干燥室压强和加热板温度对冻干时间和能耗影响显著，并呈二次函数规律变化，在本试验条件下影响的主次因素依次为：物料厚度、干燥室压强、加热板温度。对单位厚度冻干时间进行岭嵴分析，给出了在本试验因素取值范围内的冻干过程优化参数：物料厚度为9mm；干燥室压强为50Pa；加热板温度为39℃，优化的物料单位厚度冻干时间为0．41h。     

“一种鳗弧菌二价疫苗及其制备和使用方法”获国家发明专利授权

正日前,由中国水产科学研究院黄海水产研究所莫照兰研究员等人发明的"一种鳗弧菌二价疫苗及其制备和使用方法"获国家发明专利授权,专利号为:ZL2013101632606。此发明专利在多年流行病学调查的基础上,针对引起我国水产养殖动物弧菌病的病原鳗弧菌(Vibrio anguillarum),进行了灭活疫苗的制备和开发。将鳗弧菌O1血清型和O2血清型两株菌分别活化、发酵培养和甲醛灭活后,菌悬液按细胞数     

牙鲆抗鳗弧菌病家系筛选及其分析

2007年以从养殖牙鲆(Paralichthys olivaceus)中筛选出的抗病选育群体、从日本引进的日本群体以及从黄海捕捞的野生群体为基础,进行各种组合方式的交配,建立了63个牙鲆家系;2008年又建立了30个家系。对2007年培育的59个家系和2008年培育的30个家系进行了鳗弧菌(Vibrio anguillarum)感染实验,结果表明,不同家系间的抗病力存在极显著的差异(P0.01)。从2007年培育的59个家系中筛选出4个抗病家系,鳗弧菌感染后存活率达到50%以上;2008年的30个家系中5个家系抗病力较强,鳗弧菌感染后的存活率达到60%以上。2008年培育的30个家系其4月龄和6月龄的感染后存活率的相关系数为0.403(P0.05),表明不同生长时期的感染结果具有一致性。230d的体长和体质量与对鳗弧菌的抗病力存在显著的相关关系(P0.05),皮尔森相关系数分别为0.282和0.237。研究发现,日本群体与抗病选育群体的杂交后代抗病性能表现优异,可以通过日本群体与中国群体间的杂交进行遗传改良,达到培育出高产、抗病牙鲆新品种的目的。     

3株O3血清型鳗弧菌灭活疫苗的免疫原性和免疫保护效果

鳗弧菌(Vibrio anguillarum)O3血清型菌株是感染鱼类的重要病原菌,本文研究了3株O3血清型鳗弧菌(SMP1、SMP3和SMP4)灭活疫苗的免疫原性和免疫保护。首先在牙鲆(Paralichthys olivaceus)体内对3株鳗弧菌进行复壮;检测复壮后的菌株毒力,检测的3株鳗弧菌对蓝蔓龙(Trichogaser trichopterus)的半数致死量(LD50)分别为105.1 CFU/ml(SMP1)、104.7 CFU/ml(SMP3)和105.4 CFU/ml(SMP4);制备了3株菌的甲醛灭活疫苗,注射免疫牙鲆,免疫后第7天可检测到牙鲆的血清特异性抗体产生,免疫后第28天的血清特异性抗体效价为1:1280(SMP1)、1:640(SMP3)和1:905(SMP4),提供的免疫保护率(Relative percent survival rate,RPS)为94.4%(SMP1)、100%(SMP3)和73.7%(SMP4)。研究表明,3株致病性O3血清型鳗弧菌菌株具有良好的免疫原性,其中SMP3为最适疫苗候选株。本研究为鳗弧菌O3血清型疫苗的开发应用奠定了基础。     

Different approaches to expressing Edwardsiella tarda antigen GAPDH in attenuated Vibrio anguillarum for multivalent fish vaccines

With the development of gene technology, expressing heterologous antigens in attenuated bacteria has become an important strategy to design multivalent vaccines. In our previous work, an attenuated Vibrio anguillarum named MVAV6203 was developed and proven to be an efficient live vaccine candidate. In this research, we aimed to express protective antigen glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) of Edwardsiella tarda in attenuated Vibrio anguillarum to establish a multivalent V. anguillarum vector vaccine. Several strategies were compared between low‐ vs. high‐copy plasmid‐mediated antigen expression, in vivo‐inducible vs. constitutive antigen expression and intracellular vs. surface‐displaying antigen expression. Zebrafish, Danio rerio (Hamilton), was applied as the fish model to evaluate the immune protection of the V. anguillarum vector vaccine candidates. Our results demonstrated that V. anguillarum MVAV6203 (pUTatLNG40), which harbours a low‐copy plasmid‐loaded antigen surface display system under the control of a constitutive promoter, presented the best protective efficacy against the infection of Vibrio anguillarum (relative per cent survival, RPS = 85%) and Edwardsiella tarda (RPS = 70%).     

Secretory expression of recombinant proteins in an attenuated Vibrio anguillarum strain for potential use in vaccines

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.     

Surface-exposed expression of Edwardsiella tarda EseB in live attenuated Vibrio anguillarum based on novel surface display systems

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.     

Gene Cloning and Expression of Major Capsid Protein of Large Yellow Croaker Iridovirus and Its Secretion Based on α‐hemolysin Transport System

Large yellow croaker iridovirus (LYCIV) is an important pathogen of mariculture fish. The complete gene of an important protective antigen, major capsid protein (MCP), of LYCIV was amplified by polymerase chain reaction and was cloned into pET‐16b for expression in Escherichia coli. The MCP‐encoded gene was in‐frame fused to the E. coliα‐hemolysin transport elements to construct the MCP secretory expression plasmid pMOhlyM, and its secretion characters in E. coli and attenuated Vibrio anguillarum MVAV6203 were investigated. With the aid of E. coliα‐hemolysin transport system, about 400 μg/L MCP was secreted into culture supernatant in E. coli, while intracellularly expressed MCP in MVAV6203 was not secreted. The study provides the foundation for attempts to secrete viral‐originated antigens by the E. coli HlyA transporter in bacterial hosts and to explore feasible ways to develop multivalent live vaccines against both bacterial and viral pathogens.     

Modification of the hyperosmotic infiltration method of vaccination against vibriosis in cultured ayu, Plecoglossus altivelis Temminck and Schlegel

Abstract. A vaccine solution of a formalin-killed culture of Vibrio anguillarum cells was observed to be toxic to young ayu when administered by the hyperosmotic infiltration method. The toxin was present in the culture broth. After the toxin was removed from the broth by centrifugation, the fish were dipped in 5.32% NaCl solution for 2 min and then in a solution containing precipitated cells for 3 min. The immunized fish were protected against vibriosis when challenged one month after immersion. The bacterin was administered to ayu by a further two methods, both using lyophilized whole cells of formalin-killed V. anguillarum. In one method, the fish were placed in a 5.32% NaCl solution for 2 min and then in a solution containing lyophilized cells at 2 g/l of well water for 3 min (two-step immersion). In the other method, the fish were placed in a 5.32% NaCl solution containing lyophilized cells also at 2 g/l for 3 min (one-step immersion). A high level of protection against artificial challenge was achieved with either method. No agglutinating antibodies to V. anguillarum were detected in either the serum or mucus of fish dipped in a vaccine solution, a supernatant, or a precipitated solution, one month after immersion. On the other hand, serum titres were detected in fish vaccinated by injection, although no titres were detected in mucus. LD₅₀ values are presented for the virulence of the V. anguillarum strain. Compared to the original strain, virulence increased after the third passage in ayu, but decreased after the thirteenth passage in medium.     

Effect of poly-β-hydroxybutyrate on Chinese mitten crab, Eriocheir sinensis, larvae challenged with pathogenic Vibrio anguillarum

This study investigated the protective effect of poly-β-hydroxybutyrate (PHB) on Chinese mitten crab, Eriocheir sinensis, zoea larvae challenged with pathogenic Vibrio anguillarum. PHB was delivered to the crab larvae through rotifer and Artemia bioencapsulation. Zoea 3 larvae were challenged with V. anguillarum at a final concentration of 10(5) CFU mL(-1). PHB-enriched rotifers and Artemia nauplii were added to the culture water 24 h prior to, upon and 24 h after challenge. The results confirmed that PHB could enhance the survival and growth of unexposed E. sinensis larvae. Moreover, PHB protected larvae from the pathogen as the larvae fed PHB-enriched live food showed the highest survival and development rate in all challenged groups (P < 0.05). Furthermore, larval performance was the best when PHB was delivered to the larvae 24 h before challenge (P < 0.05). In conclusion, our results indicate that PHB can be used as part of an effective strategy to protect E. sinensis larvae from V. anguillarum resulting in higher survival and better growth, especially when applied before the challenge.     

Evidence for lack of antigenic competition among various combinations of Vibrio anguillarum, Yersinia ruckeri, Aeromonas salmonicida and Renibacterium salmoninarum bacterins when administered to salmonid fishes

Abstract. Bacterins of Vibrio anguillarum, Yersinia ruckeri, Aeromonas Salmonicida , and Renibacterum salmoninarum were administered alone and in combination to salmonid fishes and the level of protective immunity compared. With each pathogen, the protection obtained with monovalent with A. salmonicida bacterin alone conferred some protection against challenges with Type II V. anguillarum and Y. ruckeri. Also, the combination of A. salmonicida and R. salmoninarum bacterins appeared to potentiate the protection conferred against A. salmonicida. The potential of multivalent vaccines for protecting fish from several diseases appears to be real.     

溶藻弧菌鞭毛蛋白flaC基因DNA疫苗对红笛鲷的免疫保护研究

为研究溶藻弧菌鞭毛蛋白flaC基因DNA疫苗对红笛鲷的免疫保护作用,实验构建了重组真核表达质粒pcDNA-flaC并将该质粒肌肉注射红笛鲷,采用PCR、RT-PCR、ELISA和攻毒试验等方法检测了该真核表达质粒在红笛鲷组织内的分布、表达和对红笛鲷的免疫保护.PCR结果显示,免疫接种7和28 d,注射点周围肌肉、鳃、肾脏、肝脏和脾脏都存在质粒分布；RT-PCR结果显示,免疫接种后第7天、14天和28天,红笛鲷不同组织内均有目的基因表达.ELISA结果表明,鱼血清内产生了抗FlaC蛋白的抗体,表明DNA疫苗免疫后鱼体表达了目的蛋白,并诱导产生了相应抗体.攻毒实验表明,免疫后的红笛鲷能较好地抵抗致病性溶藻弧菌的感染.结果表明,质粒pcDNA-flaC可能是抵抗溶藻弧菌感染的有效的疫苗候选物.     

溶藻弧菌外膜蛋白（Va-OMP）的免疫原性及免疫保护性

利用初步制备的溶藻弧菌外膜蛋白（outer membrane protein of Vibrio alginolyticus, Va-OMP）、溶藻弧菌蛋白分子量58 kD外膜蛋白（Va-OMP58）和溶藻弧菌（Vibrio alginolyticus, Va）灭活菌苗、溶藻弧菌脂多糖（lipopolysaccharides of Vibrio alginolyticus, Va-LPS）菌苗等进行主动保护性和被动保护性的实验比较，VaOMP、Va-OMP58免疫组小鼠免疫后用活菌攻击，免疫保护率为62.5%～86.7%,而Va-LPS组和Va灭活菌苗组的免疫保护率为50%～62.5%，对照组的免疫保护率仅为6.7%。用不同抗血清免疫小鼠，活菌攻击后，Va-OMP组存活率达到53.3%和66.7%，VaOMP58组次之，存活率为40%和50%，Va灭活菌组的存活率为33.3%和46.7%，对照组为13.3%。与对照组相比较，Va-OMP组差异较显著，保护效果较好。溶藻弧菌外膜蛋白和蛋白分子量58 kD外膜蛋白的保护性效果高于溶藻弧菌灭活菌苗和溶藻弧菌脂多糖的保护性效果，证明外膜蛋白（outer membrane protein, OMP）具有较强的免疫原性。迟发性超敏反应试验也证明，OMP能诱发变态反应，间接证明OMP具有较强的引起细胞介导免疫反应的能力。因此实验证明，Va-OMP、Va-OMP⁵⁸是能在小鼠产生体液免疫和细胞介导免疫的保护性抗原。