口虾蛄主要过敏原原肌球蛋白的免疫活性

原肌球蛋白(TM)是甲壳类动物的主要过敏原,其氨基酸序列具有很高的保守性。本研究以口虾蛄为研究对象,旨在探明TM是否是其主要过敏原并对其相关性质进行研究。利用过敏患者血清通过免疫印迹法确定口虾蛄的主要过敏原分子量约为36ku。通过制备丙酮粉、等电点沉淀、硫酸铵盐析及加热等方法纯化了该主要过敏原。采用抗中华绒螯蟹TM多克隆抗体进行免疫学性质分析表明该蛋白为TM。抑制性免疫印迹和抑制性ELISA实验表明口虾蛄TM与凡纳滨对虾和锯缘青蟹等甲壳类动物的TM具有较强的免疫交叉反应,但与贝类动物杂色蛤的免疫交叉性较低。通过ELISA方法对甲壳类动物的TM进行定量测定,结果表明,口虾蛄肌肉中TM的含量约只有凡纳滨对虾TM含量的1/45,但仍具有一定的致敏性。     

加工方式对刀额新对虾主要过敏原免疫原性的影响

选取冻干、烘干、虾丸化以及油炸4种加工方式处理刀额新对虾,通过检测可溶性蛋白、主要过敏原含量和免疫活性的变化以及体外模拟胃液消化实验,分析加工方式对虾类过敏原免疫活性及抗酶解能力的影响。结果表明经过不同的加工处理后,分子量为35 ku左右的主要过敏原蛋白仍然存在,免疫印迹显示其免疫活性有不同程度的下降,其中虾丸化处理对免疫活性的降低程度最大,达87%,冻干处理的影响最小,仅达6.8%;而主要过敏原蛋白抗酶解能力变化不明显。结论:不同的加工方式对虾过敏原活性的降低有比较大的差异,但对于抗酶解能力影响不大。     

中国明对虾主要过敏原基因分子克隆与序列分析

就中国明对虾主要过敏原原肌球蛋白基因进行了分子克隆与序列分析.从中国明对虾肌肉中提取总RNA,反转录合成第一链cDNA;根据原肌球蛋白cDNA的保守序列设计引物并进行PCR扩增,最后测序获得了中国明对虾原肌球蛋白的cDNA序列(GenBank accession:GU233303).该cDNA 序列含有长855nt的完...     

青岛地区常见鱼类过敏原的鉴定及其交叉反应研究

选择青岛地区喜食的阿拉斯加狭鳕、蓝点马鲛、大黄鱼、真鲷、鲑、大菱鲆、鲤 等7种鱼为研究对象,利用鱼类过敏患者血清,通过SDS-PAGE及免疫印迹方法对每种鱼的过敏原蛋白进行鉴定。利用兔抗鲅鱼小清蛋白多克隆抗体,通过间接ELISA和抑制性ELISA等对7种鱼的小清蛋白之间的交叉反应进行探讨。结果表明,青岛地区居民鱼类过敏原主要为48~57、33~41、28 及17 ku的蛋白,和传统公认的小清蛋白为主要过敏原的共识不同。而针对小清蛋白的交叉反应研究也表明,不同鱼类蛋白之间存在强烈的交叉反应。因此,虽然鱼类过敏原可能不同,但如果对以上7种鱼中的一种鱼过敏,最好不要食用其他任何一种鱼。     

不同热加工方式对刀额新对虾过敏原活性的影响

选取煮(100 ℃)、蒸(100 ℃)、高压(121 ℃,0.1 MPa)这3种常见热加工方法,分别处理刀额新对虾不同的时间,通过检测总可溶性蛋白、主要过敏原含量和免疫活性的变化,分析3种热加工方式对虾类过敏原免疫活性的影响,并通过质地剖面分析,研究虾肉经过处理后组织结构的变化。经过3种热处理后,分子量为36 ku的主要过敏原蛋白仍然存在,免疫印迹的结果显示其免疫活性有不同程度的下降,高压对免疫活性的降低程度最大,30 min时免疫活性降低了97%;在25~35 ku区域出现一条新的IgE结合蛋白。质构分析结果显示,经过高压热处理后,虾肉的硬度和咀嚼度都有很大程度的降低,口感变差。结果表明:热高压处理在降低虾过敏活性的同时,对其口感也有一定的影响,有必要通过优化高温高压处理工艺,在保持虾口感及营养的基础上,降低其过敏活性。     

南极磷虾主要过敏原的分离纯化及性质研究

为了探究南极磷虾致敏问题,从南极磷虾中筛选、鉴定、分离纯化其主要过敏原,并对过敏原的性质进行研究,实验通过缓冲盐溶液提取南极磷虾蛋白;采用SDS-PAGE和Western blot(WB)筛选南极磷虾过敏原;使用高效液相色谱-串联质谱鉴定过敏蛋白;通过等电点沉淀、硫酸铵盐析、阴离子交换柱层析分离纯化过敏原;采用SDS-...     

日本囊对虾原肌球蛋白的原核表达、抗体制备以及组织表达分析

食物过敏是人类常见的一种过敏性疾病,主要由食物中的蛋白质引起。原肌球蛋白(tropomyosin,TPM)是食用虾蟹等甲壳动物引起过敏反应的主要致敏物质。研究从日本囊对虾肌肉组织中克隆了TPM基因,进行了原核表达和蛋白质纯化,并进一步制备了相应的抗体。Western-blotting分析表明原肌球蛋白在日本囊对虾组织中普遍表达,并且肌肉组织中表达量最高而鳃和皮肤组织中表达量最低。研究结果为对虾过敏性疾病的诊断和治疗以及脱敏食品开发等奠定了理论基础。     

冻融循环过程中虾过敏原的免疫原性变化

采用凡纳滨对虾为研究对象,分别将过敏原提取物在-3,-20和-80 ℃的贮存条件下反复冻融1~5次,通过测定每次冻融后的蛋白浓度,过敏原免疫原性等探讨过敏原在不同温度循环下的变化,分析冻融循环对过敏原提取物活性的影响。结果表明,虾过敏原提取物在-80、-20、-3 ℃冻融循环5次时,蛋白浓度分别下降33.1%、30.0%和24.2%;在-80和-20 ℃冻融循环4次时,主要过敏原条带发生降解,-3 ℃冻融循环5次,主要过敏原条带不会发生变化。在-80和-20 ℃冻融循环5次以及在-3 ℃冻融3次时,过敏活性降低;在-80和-20 ℃冻融5次,主要过敏原的免疫印迹条带消失,在-3 ℃冻融的免疫印迹条带没有变化。因此,应尽量减少过敏原蛋白冻融次数,保证贮存在-80和-20 ℃的过敏原冻融循环4次以内以及-3 ℃冻融循环2次以内,以保证实验结果的准确性。     

水产品过敏原危害性评价及管理建议

水产品过敏是常见的食物过敏之一。伴随着水产品消费量的增加,由食用水产品引起过敏的患者数量出现了递增趋势。为了避免水产品过敏原在加工过程中发生交叉污染,水产品企业纷纷在危害分析及关键控制点(HACCP)中增加了对过敏原的控制措施。行业主管部门也相继规范了水产品过敏原的安全管理措施以保障敏感消费者的食用安全。文章主要从水产品中主要过敏原对消费者健康的影响、过敏原的来源、过敏原的评价方法等方面对水产品中过敏原的危害性进行了分析。在此基础上,建议从完善检测技术、管理体系,加强信息管理、风险评估以及提高消费者认知等方面,推进水产品过敏原的食用安全管理工作。     

甲壳类水产品主要过敏原原肌球蛋白胶体金免疫层析快速检测方法的建立及应用

为实现甲壳类水产品主要过敏原的快速检测,实验使用凡纳滨对虾原肌球蛋白(TM)作为检测靶标,构建了胶体金免疫层析检测方法。使用天然TM分别免疫SD大鼠和新西兰大白兔制备多克隆抗体,以40 nm胶体金标记的鼠多抗作为检测抗体,以兔多抗为捕获抗体,基于双抗体夹心原理组装免疫层析试纸条,并应用于市售食物样品的检测。结果显示,组建的免疫层析试纸条可在5 min之内显示结果,视觉检出限为10 ng/mL,对虾、蟹、克氏原螯虾及美洲螯龙虾等甲壳类水产品的特异性高,但与蛤蜊、扇贝存在微弱的交叉反应;在不含甲壳类水产品的市售食品中的加标回收率为81%～126%,准确性良好;试纸条的批内、批间变异系数低于15%,市售实际食物样品的检测结果与食物过敏原标签一致。研究表明,该方法具有高灵敏度与检测特异性,可在多种基质与商业化食品中实现甲壳类过敏原的有效检测,具有较强的实际应用价值。     

Association between the interannual variation in the oceanic environment and catch rates of bigeye tuna (Thunnus obesus) in the Atlantic Ocean

The environmental processes associated with variability in the catch rates of bigeye tuna in the Atlantic Ocean are largely unexplored. This study used generalized additive models (GAMs) fitted to Taiwanese longline fishery data from 1990 to 2009 and investigated the association between environmental variables and catch rates to identify the processes influencing bigeye tuna distribution in the Atlantic Ocean. The present findings reveal that the year (temporal factor), latitude and longitude (spatial factors), and major regular longline target species of albacore catches are significant for the standardization of bigeye tuna catch rates in the Atlantic Ocean. The standardized catch rates and distribution of bigeye tuna were found to be related to environmental and climatic variation. The model selection processes showed that the selected GAMs explained 70% of the cumulative deviance in the entire Atlantic Ocean. Regarding environmental factors, the depth of the 20 degree isotherm (D20) substantially contributed to the explained deviance; other important factors were sea surface temperature (SST) and sea surface height deviation (SSHD). The potential fishing grounds were observed with SSTs of 22–28°C, a D20 shallower than 150 m and negative SSHDs in the Atlantic Ocean. The higher predicted catch rates were increased in the positive northern tropical Atlantic and negative North Atlantic Oscillation events with a higher SST and shallow D20, suggesting that climatic oscillations affect the population abundance and distribution of bigeye tuna.     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Growth performance,digestive enzyme activity and immune response of Japanese sea bass,Lateolabrax japonicus fed with fructooligosaccharide

In this experiment, a feeding trial was performed to determine the effects of fructooligosaccharide (FOS) on growth performance, digestive enzyme activity and immune response of Japanese sea bass, Lateolabrax japonicus juveniles (initial weight 38.3 ± 0.5 g), and the fish were examined following feeding with six levels of FOS (0, 0.5, 1, 2, 4 and 6 g/kg) for 28 days. Significant enhancement of weight gain (WG) and specific growth rate (SGR) was found in fish fed 1 g/kg FOS incorporated diets (p < .05), while the feed conversion ratio (FCR) in the 1, 2 g/kg FOS groups reduced significantly compared with the control (p < .05). Besides, the crude lipid in the 4, 6 g/kg FOS groups increased significantly compared with the control (p < .05). On the other hand, the erepsin and lipase activities significantly elevated in intestine of fish fed 2 g/kg FOS (p < .05) and the lysozyme activity in serum of fish fed 2 g/kg FOS were significantly higher than that in the control (p < .05). Moreover, the alkaline phosphatase activities in serum of fish fed 0.5, 1, 2 g/kg FOS were significantly higher than in control (p < .05). Regression analysis showed that the relationships between dietary FOS levels and either SGR, FCR, erepsin or lysozyme activities were best expressed by regression equations, and the optimal inclusion levels are 1.37, 1.80, 3.06, 3.11, 1.93 and 1.80 g/kg for SGR, FCR, erepsin, lipase, lysozyme and total superoxide dismutase activities, respectively. Overall, this study revealed that FOS incorporated diets could beneficial for L. japonicus culture in terms of increasing the growth, digestion and immune activities. Under the present experimental condition, the optimal supplementary level of FOS in the diet of L. japonicus is 1–3 g/kg.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Protein and amino acid composition from the mantle of juvenile O ctopus vulgaris exposed to prolonged starvation

Protein and amino acid composition of the mantle of juvenile O ctopus vulgaris (Cuvier, 1797) during fasting for 27 days were determined. Average protein content of octopus mantle was of 711.19 ± 46.80 g kg^?1 DW, and it decreased with increasing fasting days. The non‐essential amino acids content was higher (486.18 ± 11.08 g kg^?1 protein) than essential amino acids (425.82 ± 9.15 g kg^?1 protein) at the start of the experiment (unstarved animals). The results suggest that the amino acid profile of the mantle where the most abundant amino acids are Arg, His, Lys, Gly, Leu and Pro could indicate a prolonged fasting condition (>20 days) or poor nutrition of O . vulgaris. This study supports the idea of using mantle for metabolic needs of starved O . vulgaris suggesting that the degradation pathway of amino acids to pyruvate and tricarboxylic acid cycle intermediates was favoured contrary to the degradation pathway of ketogenic amino acids. Special considerations should be taken concerning Thr, Ile, Ser, Ala, Asx (Asp, Asn), Glx (Glu, Gln) (because of their fast intake) and Lys and His (due to their stable contents) during a prolonged period of fasting.     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.