鳗弧菌灭活疫苗对海水养殖大菱鲆的免疫预防研究

福尔马林灭活的鳗弧菌疫苗分别以浸泡、注射接种的方式对海水养殖大菱鲆鱼苗进行免疫 ,定期检测血清中特异性抗体。 4周后用鳗弧菌悬液 (3.7× 1 0 8cfu/ml) 0 .2ml腹腔注射感染免疫鱼。结果表明 ,灭活鳗弧菌疫苗用 2种方式免疫后都能刺激大菱鲜产生特异性免疫反应。其中注射免疫组在 1周后即可产生特异抗体 ,在 6周内一直呈上升趋势 ,最高达 1 2 2 8.8,免疫 4周后人工感染后的免疫保护力为 91 .70 %。浸泡免疫组在 3周后才产生抗体凝集价 ,凝集抗体效价最高 36 ,免疫保护力为 33.30 %。     

脂质体对牙鲆淋巴囊肿病毒核酸疫苗免疫活性的影响

将淋巴囊肿病毒核酸疫苗pEGFP-N2-LCDV0.6kb用脂质体梭华-SoftastTM包裹后肌肉注射入牙鲆体内,通过测定牙鲆外周血、肠、脾脏和前肾的淋巴细胞增殖反应、呼吸爆发活性以及抗体产生水平,评价脂质体对pEGFP-N2-LCDV0.6kb免疫活性的影响.结果表明,脂质体可显著增强该核酸疫苗的免疫活性和提高保护率,可作为一种有应用前景的核酸疫苗佐剂.     

联用抗口蹄疫病毒重组多肽及DNA疫苗诱发豚鼠产生特异免疫应答

选择一株O型口蹄病毒（FMDV）外壳蛋白VPI中21－40位及141－160位氨基酸残基两个抗原表位,组成串联结构141－160（20AA）－21－40（20AA）－141－160（20AA）,并与大肠杆菌β－半乳糖苷酶相连,在大肠杆菌中表达了此融合蛋白。同时将编码此融合蛋白的基因克隆进真核表达载体中,构建成重组表达质粒pCDM8FZ1。将pCDM8FZ1DNA与融合蛋白混合,免疫豚鼠一次,观察豚鼠产生免疫应答情况。结果显示,豚鼠可产生抗FMDV中和抗体及特异性T细胞增殖反应,部分豚鼠能抵抗FMDV的攻击,保护率为50％,证明将DNA疫苗与重组多肽疫苗结合起来作一次免疫,是可望用于预防FMDV感染的新途径,值得进一步探索。     

从鳗弧菌M3 Fosmid文库筛选aroA基因

为克隆鳗弧菌aroA基因 (它在细菌中编码合成芳香族氨基酸的关键酶--5-烯醇氏丙酮酰莽草酸3-磷酸(EPSP)合成酶),在实验室中构建了鳗弧菌M3大分子量基因组Fosmid文库,并通过克隆测序获得了部分序列,以此为基础,通过延伸测序获得了ar oA基因序列全长. 该基因全长1281bp,编码427个氨基酸,包含一个11个氨基酸残基的信号肽;编码区GC平均含量为46.8%,推测分子量为46177 Da.相似性分析表明,鳗弧菌的aroA核苷酸和氨基酸序列与其他弧菌的相似性最高,分别在73%～75%和78%～82%;进化树结果也表明鳗弧菌ar oA与其他弧菌的亲缘关系最近.aroA基因的克隆为构建鳗弧菌弱毒疫苗奠定了基础.     

大菱鲆(Scophthalmus maximus)一种CXC趋化因子的克隆及其表达特征

从大菱鲆脾脏cDNA文库中筛选到一种CXC趋化因子。它的全长cDNA含有一个81bp的5’UTR,一个414bp的开放阅读框和一个449bp的3’UTR。开放阅读框编码了137个氨基酸残基。经过PCR扩增发现,该CXC趋化因子基因内含有4个外显子和3个内含子。RT-PCR分析表明,大菱鲆CXC趋化因子在正常脾脏和头肾中表达强烈;在胚胎的不同发育时期,大菱鲆CXC趋化因子从体节期开始才有微弱的表达。大菱鲆胚胎细胞(TEC)在用鳗弧菌感染后6h也有强烈的表达。这些结果表明该CXC趋化因子在大菱鲆免疫应答中起着重要作用。     

大菱鲆病原鳗弧菌生物学及分子特征研究

从山东沿海养殖发病大菱鲆分离了5株鳗弧菌。5株菌对大菱鲆有较强的致病性,LD50范围为2.32×101～4.03×102cfu/g鱼;5株菌分别属于O1、O2、O3血清型,菌株之间的16SrRNA基因序列相似性范围97.1%～99.9%;与国外报道的鳗弧菌的序列相似性范围为89.7%～99.9%。5株菌的生理生化特征与鳗弧菌标准菌株一致,能产生多种胞外酶和溶血素;都含有金属蛋白酶基因和溶血素基因,其中3株菌各含有一个67kb大质粒。病原菌对青霉素、苯唑青霉素、氨苄青霉素、先锋霉素Ⅳ、先锋霉素Ⅴ、先锋霉素Ⅵ、链霉素、强力霉素、洁霉素、万古霉素、灭滴灵等11种常用抗菌药物有抗性,只对新生霉素、呋喃妥因、利福平、新霉素等4种药物敏感。     

鲆鲽类主要病原菌抗血清的制备及应用

用甲醛灭活8种鲆鲽类主要病原菌,鳗弧菌(Vibrio anguillarum)、创伤弧菌(Vibrio vulnificus)、哈维弧菌(Vibrio harveyi)、溶藻弧菌(Vibrio alginolyticus)、嗜水气单胞菌(Aeromonas hydrophila)、豚鼠气单胞菌(Aeromonas caviae)、迟缓爱德华菌(Edwardsiella tarda)、鮰爱德华菌(Edwardsiella ictaluri),制备成灭活疫苗后肌肉注射大菱鲆获得各病原菌抗血清。结果显示,抗鳗弧菌血清、抗哈维弧菌血清、抗豚鼠气单胞菌血清、抗鮰爱德华菌效价为1:6 400,抗创伤弧菌血清、抗溶藻弧菌血清、抗嗜水气单胞菌血清效价为1:12 800,抗迟缓爱德华菌血清效价为1:102 400。应用ELISA分析各抗血清与8种病原菌及迟缓爱德华菌蛋白的免疫交叉反应,结果显示,各病原菌与其本身的抗血清反应最为强烈,而与其他抗血清间有程度不等的交叉反应;菌蛋白与其相应的菌抗血清反应最为强烈,与其他病原菌抗血清反应较弱。本研究所制备的病原菌抗血清效价较高,且具有特异性,可应用于鲆鲽类病原菌的检测。     

鲈鱼口服生物胶囊疫苗的研究

以暴发弧菌病的鲈鱼 (Lateolabraxjaponicus)鱼苗体内分离的 1株病原菌W 1(Vibrioanguillarum)为材料 ,分别以直接浸浴法、创伤后浸浴法和肌肉注射法人工感染鲈鱼鱼苗。结果表明 ,这三种感染方式均能使鲈鱼发病 ,其半致死浓度 (LD50 )分别为2 75× 10 7cfu/ml ,4 .68× 10 6cfu/ml和 3.72× 10 5cfu/尾。在此基础上 ,分别以福尔马林灭活法和喷雾干燥法制备了全细胞疫苗和微胶囊疫苗。将全细胞疫苗及微胶囊疫苗以直接拌入饵料法和卤虫携带法等两种方法口服免疫接种鲈鱼鱼苗 ,一周后以W 1活菌攻毒( 2 50× 10 6cfu/尾 )。结果表明 ,各免疫组一周的累积死亡率远低于两组对照组 ( 95% )。其中 ,以卤虫携带的生物微胶囊疫苗组的一周累积死亡率最低 ,为 2 5.0 % ;以卤虫携带的生物全细胞疫苗组的一周累积死亡率较高 ,为 60 .0 %。因而 ,以卤虫携带的生物胶囊疫苗组的免疫保护力最高 ,为 73.7% ,其次为微胶囊疫苗直接投喂组 ,保护力为 56.8%。直接投喂和卤虫携带法的全细胞疫苗也显示了一定的免疫效果 ,其免疫保护力分别为 52 .6%和 36.8%。     

免疫亲和分离膜研究进展

免疫亲和膜是基于抗原和抗体间高特异性可逆相互作用对抗体或抗原进行分离的一项新型分离技术,兼有膜分离和亲和色谱的优点.介绍了免疫亲和色谱的作用机理、膜材料和配体的选择,以及免疫亲和膜在抗体分离纯化和快速诊断测试方面的应用,同时还对免疫亲和膜的未来发展进行了展望.     

大菱鲆疾病早期快速检测方法——胶体金免疫层析试纸的研制与建立

使用成分单一的牛血清白蛋白(BSA)为模拟病原,以胶体金标记兔抗血清(即大菱鲆免疫球蛋白多抗)作为检测示踪物,并分别将BSA和葡萄球菌A蛋白印记到硝酸纤维素膜上制成检测线和对照线,通过一系列工艺创制与组装配套,首次成功制备了一套完整的大菱鲆抗体快速检测试纸。采用大菱鲆抗BSA血清作为阳性样本,以健康大菱鲆血清作为阴性样本,用以检验试纸的性能,并与酶联免疫吸附实验(ELISA)法检测结果相比较。结果表明:本试纸检测抗体的特异性与敏感性均很高,与ELISA方法相当,而且使用方便,不需专业技能和额外的试剂与辅助仪器设备,5 min内即可用裸眼获得观察结果,很适合于基层生产操作及户外调研使用。以该实验为基础建立起来的抗体检测试纸,亦可推广应用于其他病害抗体的检测,可为鱼类疾病早期发生提供简易、快捷和操作性强的诊断方法。     

Assembly of Immunogenic Protein Particles toward Advanced Synthetic Vaccines

Immunogenic carrier proteins such as the non-toxic diphtheria toxin variant, cross-reacting material 197 (CRM197), are widely used in subunit vaccine formulations to boost immunogenicity of chemically conjugated antigens. Conjugate vaccines are inherently expensive due to laborious manufacturing steps. Here, this work develops a particulate vaccine platform based on using engineered Escherichia coli to assemble CRM197-antigen fusion proteins into discrete submicron-sized particles. This approach enables precise loading of diverse antigens and epitopes enhancing their immunogenicity. A cost-effective, high-yield, and scalable biomanufacturing process is developed. Purified particulate CRM197-antigen vaccines are ambient-temperature stable. CRM197 particles incorporating pathogen-specific antigens or epitopes from SARS-CoV-2, Streptococcus pyogenes (group A), and Mycobacterium tuberculosis induced cell-mediated and humoral immune responses mediating protective immunity in respective animal models of infection. The CRM197 particle vaccine platform is versatile, enabling co-delivery of selected antigens/epitopes together with immunogenic CRM197 as discrete stable particles avoiding laborious manufacture of soluble CRM197 and antigen followed by chemical conjugation.     

重组质粒pcFlaA在青石斑鱼肌肉中的表达研究

在哈维氏弧菌TS-628菌株鞭毛丝蛋白FlaA基因末端加上一段编码Flag短肽的核苷酸序列作为检测标记后,将该基因克隆到真核表达载体pcDNA3.1( ),酶切、PCR扩增及重组质粒测序证实基因片段插入正确,将该重组质粒命名为pcFlaA.将pcFlaA以肌肉注射方式免疫青石斑鱼.免疫后第7天开始检测鞭毛丝蛋白在石斑鱼肌肉中的表达状况,之后每隔1周检测1次,共检测4次.首先采用PCR技术在DNA水平检测重组质粒转染石斑鱼肌肉细胞的情况,再以RT-PCR法在mRNA水平上检测转染质粒在鱼肌肉中的转录,最后以免疫组化染色技术在蛋白质水平上检测目的蛋白的表达.结果在DNA及mRNA水平上均可检测到目的条带,在蛋白质水平上可检测到明显阳性位点,由此证实pcFlaA可以转染石斑鱼肌肉细胞并可在其中进行表达,而且质粒在鱼体内持续表达的时间至少1个月.     

Distribution of vaccine/antivirals and the ‘least spread line’ in a stratified population

We describe a prioritization scheme for an allocation of a sizeable quantity of vaccine or antivirals in a stratified population. The scheme builds on an optimal strategy for reducing the epidemic''s initial growth rate in a stratified mass-action model. The strategy is tested on the EpiSims network describing interactions and influenza dynamics in the population of Utah, where the stratification we have chosen is by age (0–6, 7–13, 14–18, adults). No prior immunity information is available, thus everyone is assumed to be susceptible—this may be relevant, possibly with the exception of persons over 50, to the 2009 H1N1 influenza outbreak. We have found that the top priority in an allocation of a sizeable quantity of seasonal influenza vaccinations goes to young children (0–6), followed by teens (14–18), then children (7–13), with the adult share being quite low. These results, which rely on the structure of the EpiSims network, are compared with the current influenza vaccination coverage levels in the US population.     

水产养殖动物核酸疫苗的研究与应用现状

介绍了鱼用核酸疫苗载体的构建、诱导的免疫反应机制、接种途径、佐剂的免疫促进作用及安全性等研究现状。     

激光光散射法表征人用皮卡狂犬病疫苗分子量与人体副反应关系的观察

为了解人用皮卡狂犬病疫苗分子量与人体副反应的关系，用激光光散射法进行了实验。结果表明：当疫苗中皮卡浓度为１１．９５ｍ ｇ／ｍ ｌ时，其重均分子量Ｍ ｗ ＝ ２９．６×１０４，２０名志愿者中７人出现局部和全身过敏反应；当疫苗中皮卡浓度为１ｍ ｇ／ｍ ｌ时，其重均分子量Ｍｗ ＝ ５．６×１０４，２０名志愿者中无一人出现接种后副反应。这说明，用激光光散射法检测人用皮卡狂犬病疫苗分子量的大小与人体副反应的关系密切。     

Applications of polymeric adjuvants in studying autoimmune responses and vaccination against infectious diseases

Polymers as an adjuvant are capable of enhancing the vaccine potential against various infectious diseases and also are being used to study the actual autoimmune responses using self-antigen(s) without involving any major immune deviation. Several natural polysaccharides and their derivatives originating from microbes and plants have been tested for their adjuvant potential. Similarly, numerous synthetic polymers including polyelectrolytes, polyesters, polyanhydrides, non-ionic block copolymers and external stimuli responsive polymers have demonstrated adjuvant capacity using different antigens. Adjuvant potential of these polymers mainly depends on their solubility, molecular weight, degree of branching and the conformation of polymeric backbone. These polymers have the ability not only to activate humoral but also cellular immune responses in the host. The depot effect, which involves slow release of antigen over a long duration of time, using different forms (particulate, solution and gel) of polymers, and enhances the co-stimulatory signals for optimal immune activation, is the underlying principle of their adjuvant properties. Possibly, polymers may also interact and activate various toll-like receptors and inflammasomes, thus involving several innate immune system players in the ensuing immune response. Biocompatibility, biodegradability, easy production and purification, and non-toxic properties of most of the polymers make them attractive candidates for substituting conventional adjuvants that have undesirable effects in the host.     

Vaccine delivery: Nanopatch‐Targeted Skin Vaccination against West Nile Virus and Chikungunya Virus in Mice (Small 16/2010)

The ‘Nanopatch’ (NP) comprises arrays of densely packed projections with a defined geometry and distribution designed to physically target vaccines directly to thousands of epidermal and dermal antigen presenting cells (APCs). These miniaturized arrays are two orders of magnitude smaller than standard needles—which deliver most vaccines—and are also much smaller than current microneedle arrays. The NP is dry‐coated with antigen, adjuvant, and/or DNA payloads. After the NP was pressed onto mouse skin, a protein payload co‐localized with 91.4 ± 4.1 APC mm⁻² (or 2925 in total) representing 52% of the delivery sites within the NP contact area, agreeing well with a probability‐based model used to guide the device design; it then substantially increases as the antigen diffuses in the skin to many more cells. APC co‐localizing with protein payloads rapidly disappears from the application area, suggesting APC migration. The NP also delivers DNA payloads leading to cutaneous expression of encoded proteins within 24 h. The efficiency of NP immunization is demonstrated using an inactivated whole chikungunya virus vaccine and a DNA‐delivered attenuated West Nile virus vaccine. The NP thus offers a needle‐free, versatile, highly effective vaccine delivery system that is potentially inexpensive and simple to use.     

DDA提高结核杆菌组合DNA疫苗免疫效果的研究

将编码结核杆菌的三种抗原Ag85B,MPT64,MPT83的基因片段分别插入到真核表达载体中,混合后作为组合疫苗免疫小鼠,DDA作为佐剂提高了三价疫苗的免疫原性和免疫保护效果.添加DDA后Ag85B,MPT64,MPT83抗原特异的IFN-γ的含量分别为(265.37±79.2)U/ml,(185.31±58.3)U/ml,(108.13±54.4)U/ml,分别比非佐剂组高16U/ml,45U/ml,2 U/ml.IL-4的含量在各组中相差不多,仅为纳克级.攻毒后细菌计数结果显示,肺脏和脾脏的载菌量在添加佐剂的三价组中降低了三个数量级,都达到了未加佐剂组相应脏器的一半菌量.病理切片显示结果与载菌量一致,添加佐剂组肺部淋巴细胞相对较少,巨噬细胞增多.因此,DDA作为佐剂提高了组合疫苗的免疫效果.