巨型艾美耳球虫偏菱形样蛋白EmRP免疫原性分析

在前期研究中,从巨型艾美耳球虫(Eimeria maxima)cDNA文库中筛选到了免疫保护性抗原偏菱形样蛋白EmRP,本研究为揭示该抗原的免疫保护机理,进一步检测了其免疫原性。以E.maxima卵囊cDNA为模板,用RT-PCR技术扩增EmRP,并构建真核表达质粒pVAX1-EmRP,将其经腿部肌肉注射2周龄的雏鸡,3周龄加强免疫。分别于首次免疫后和加强免疫后1周,采集血清,用ELISA方法检测血清特异性IgG水平,用荧光定量PCR方法(qPCR)检测细胞因子水平,用流式细胞术检测CD4^+T淋巴细胞和CD8^+T淋巴细胞的比例,分析EmRP诱导的免疫反应。序列分析表明,EmRP含有偏菱形样蛋白超家族成员保守区域,为非跨膜蛋白,分子量约为28.4 kD。真核表达质粒pVAX1-EmRP免疫鸡后,与对照组相比,鸡体IFN-γ、IL-2、IL-10和IL-17等细胞因子转录水平显著提升;CD8^+和CD4^+ T细胞比例显著提高,而血清特异性IgG水平与对照组差异不显著。结果表明,EmRP诱导的免疫保护作用主要是由其诱导的细胞免疫反应实现的,可以作为研制E.maxima新型疫苗的候选抗原。     

鸡巨型艾美耳球虫卵囊呼吸作用的研究

采用纯种巨型艾美耳球虫(Eimeria maxima)卵囊,人工感染3周龄的石歧杂雏鸡,及时收集卵囊,以0.1N硫酸溶液悬浮,用微量呼吸检压仪检测卵囊呼吸作用。试验表明:孢子化过程中最初十小时内该球虫卵囊的耗氧量为3.59μl/小时/10~6个卵囊。耗氧量的变化与其细胞分裂过程相对应,耗氧量在三次核分裂前夕降低,但在分裂期升高。最高耗氧量出现于子孢子形成期,达18.91μl/小时/10~6卵囊。孢子化后卵囊的呼吸作用较缓慢,耗氧量为0.85μl/小时/10~6个卵囊。完成孢子化所需时间为26小时。     

过饱和溶氧对大菱鲆生长及消化酶的影响

探讨工程化养殖中过饱和溶氧对大菱鲆生长的影响,通过充入液态氧与空气,使试验水体的溶氧分别达到过饱和溶氧水平(12±1)mg/L和(14±1)mg/L,正常溶氧水平(7.0±0.5)mg/L.结果显示,过饱和溶氧组的大菱鲆生长速度及饵料利用率明显高于正常溶氧组(p<0.05),而在过饱和溶氧条件下与正常溶氧条件下大菱鲆的丰满度无显著差异(P>0.05).对大菱鲆胃、肠道消化酶、脂肪酶与淀粉酶的分析发现,大菱鲆胃蛋白酶活性在过饱和溶氧条件下较正常溶氧有明显提高(p<0.05),而脂肪酶与淀粉酶的酶活力未得到显著性提高(p>0.05).     

纯氧充气对大菱鲆生长及水质指标的影响

主要研究了纯氧增氧技术对大菱鲆的生长及养殖水体中几项重要水质因子的影响。本实验中,实验池中的溶氧最低能维持在8.96±0.16mg/L,最高时可达到12.2±0.71mg/L。经过282d的培养,大菱鲆体重由30.43±0.42g达到1103.73±19.00g,日增重率平均为1.24±0.14%,整个实验期间大菱鲆均处于快速生长状态。至实验结束时,养殖密度最终达到33.99±0.59kg/m3。养殖水体中,pH变化范围在7.62～8.03之间,平均pH为7.76±0.05。COD变化范围在0.75～1.85mg/L,平均COD为1.04±0.16mg/L。亚硝酸盐浓度1.27～7.23μg/L之间,平均浓度在3.23±0.21μg/L。氨氮浓度一般维持在0.07～0.28mg/L,平均浓度为0.14±0.02mg/L。硝酸盐浓度在0.39～0.69mg/L之间,平均浓度为0.51±0.02mg/L。COD、pH、亚硝酸盐浓度很低,均在渔业水质标准所规定的范围以内。     

Purification and characterization of stomach protease from the turbot (Scophthalmus maximus L.)

Proteolytic activity in the different parts of the digestive tract of the turbot (Scophthalmus maximus L.) were studied in this work. One pure protease was isolated from turbot stomach and its behavior was studied. Results showed the optimum pH for proteases in the different parts of the digestive tract of the turbot were pH 2.0 for the stomach, pH 8.0 for the pylorus cecum, pH 8.0 for the foregut, pH 8.5 for the midgut, and pH 8.0 for the hindgut. The activity of proteases in the different parts of the digestive tract were in the sequence pylorus cecum protease > stomach protease > foregut protease > midgut protease > hindgut protease. The stomach protease was purified by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose F.F. and Sephadex G-100. The purified enzyme gave a single band in SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Its molecular weight was found to be approximately 42,000 Da. The enzyme is stable at pH 1.0–9.0 and at temperatures below 40°C. Its activity was maximum at pH 2.0 and 40°C. When reaction time was prolonged the optimum temperature of the enzyme tended to decline. The enzyme was activated by Mn²⁺ and Cu²⁺ and inactivated by Fe³⁺. It was fully inhibited by pepstatin and partially inhibited by PMSF, TPCK, PCMB, and NBS. These results imply the enzyme is a pepsin.     

氯化钴在大菱鲆饲料中的安全性评价

通过研究饲料中的氯化钴对大菱鲆幼鱼的存活、生长性能、血清生理生化指标、抗氧化酶活力、消化酶活力、血清和肝脏中VB12含量、肌肉和肝脏中钴的残留量、肝脏和肠道组织结构的影响来评估氯化钴在海水鱼饲料中的安全性。通过在基础饲料中添加氯化钴制剂来配制4种具有不同氯化钴含量(0、8、40及80 mg/kg,不计结晶水重量)的等氮等能的实验饲料,每个饲料处理设6个重复,每个重复养殖30尾大菱鲆;养殖实验为期10周。实验结果表明:饲料中添加氯化钴对试验鱼的存活没有显著影响(P0.05),但饲料中80 mg/kg的氯化钴显著降低了大菱鲆的特定生长率、终末体重、摄食率、肝体比和脏体比(P0.05);饲料中高剂量氯化钴(40和80 mg/kg)显著降低了大菱鲆血清过氧化氢酶、谷草转氨酶和碱性磷酸酶活力以及大菱鲆胃淀粉酶和胰蛋白酶活力(P0.05);大菱鲆的血糖含量、血清和肝脏中VB12含量、肝脏和肌肉中钴的残留量均随饲料中钴的添加量增加而显著升高(P0.05);饲料中添加氯化钴对大菱鲆的肠道组织结构没有产生明显影响,但饲料中添加40和80 mg/kg的氯化钴增宽了肝脏肝血窦。研究结果表明:实验条件下,氯化钴在大菱鲆饲料中的添加量8 mg/kg是安全的。     

大杯伞柄多糖与菌丝体粗多糖的免疫识别方法初探

采用热水(80℃)提取和75%乙醇沉淀、Sepharose CL-4B凝胶过滤及DEAE Sepharose CL-4B阴离子交换等方法分离纯化大杯伞(Clitocybe maxima)柄粗多糖,获得伞柄多糖纯品,制备多糖抗体;采用热水法提取大杯伞菌丝体粗多糖。用碳二亚胺法将纯化伞柄多糖与牛血清白蛋白进行共价化学偶联,并将偶联产物(拟糖蛋白)免疫兔子以制备抗血清,以酶联免疫法检测抗体滴度。依据该抗体与来自相同菌株的菌丝体粗多糖免疫反应差异对这两种多糖进行免疫识别,结果表明:经过亲和层析纯化后,第六次免疫抗血清对伞柄多糖的抗体滴度可达64K,而且对菌丝体粗多糖反应呈阴性,显示该抗体可识别这两种多糖之间的结构差异。     

Colouring ornamental fish (Cyprinus carpio and Carassius auratus) with microalgal biomass

Koi carp and goldfish value increases with intensity of skin colour, which is an important quality criterion. Fish cannot fully synthesize their own carotenoid colourings and these must therefore be included in their diet. Two trials were undertaken to investigate skin colour enhancement in ornamental species (i.e. three chromatic varieties of koi carp (Cyprinus carpio), namely Kawari (red), Showa (black and red) and Bekko (black and white) and goldfish (Carassius auratus)) by feeding a dietary carotenoid supplement of freshwater microalgal biomass [Chlorella vulgaris, Haematococcus pluvialis, and also the cyanobacterium Arthrospira maxima (Spirulina)], using a diet containing synthetic astaxanthin and a control diet with no colouring added for comparison. In the first trial, five homogeneous duplicate groups of 25 juvenile koi carp (C. carpio) (initial mean body weight 24.6 ± 0.7 g) were fed, for 10 weeks, one of the four diets containing 80 mg colouring/kg diet. In the second trial, this procedure was repeated for five homogeneous duplicate groups of 25 goldfish (C. auratus) (initial mean body weight of 0.9 ± 0.1 g). Initial and final samples of skin along the dorsal fin were withdrawn, from five fish per group, for subsequent analysis of total carotenoid content (spectrophotometric analysis), and red hue (colorimetric analysis, CIE (1976) L* a* b* colour system). Growth and feed efficiency were not significantly different between groups administered by the various dietary treatments. In both trials, dietary carotenoid supplementation increased total skin carotenoid content. The more efficient colouring for koi carps was found to be C. vulgaris biomass, providing both maximum total carotenoid deposition and red hue for the three chromatic koi carp varieties studied, and particularly for the kawari variety. For goldfish the best colouring obtained, as ascertained by total carotenoid content, was also achieved using C. vulgaris biomass, and red hue was maximum when using H. pluvialis biomass.     

Excavating sponges that are destructive to farmed pearl oysters in Western and Northern Australia

Species of the family Clionaidae (Porifera: Demospongiae) that excavate shell of the silver‐lip pearl oyster, Pinctada maxima, in north Western and Northern Australia are described. Two species belong to the genus Cliona and one species to the closely related genus Pione. Cliona orientalis has only recently been reported from Australia in living and dead coral on the Great Barrier Reef, and this is the first report of this species from north Western and Northern Australia. Cliona dissimilis is reported from Australia for the first time. Pione velans was first described from Shark Bay, Western Australia and this study extends its distribution from Albany, south Western Australia to Port Bremer in the Northern Territory. The most common species found was C. dissimilis. Pione velans and C. orientalis were also present, although the latter species was rare. Cliona dissimilis and P. velans were found to be sexually reproductive in some shells with specimens of C. dissimilis with oocytes in May and P. velans in September 1999. Egg development was synchronous, indicating that the sponges were oviparous and would broadcast gametes in a spawning event.     

Growth of cultured mussels (Perna canaliculus Gmelin 1791) at a deep-water chlorophyll maximum layer

We tested the hypothesis that yield of the mussel Perna canaliculus (Gmelin 1791) could be increased by placing culture ropes deep into the water column to take advantage of deep‐water chlorophyll a maxima. The study site, in Pelorus Sound, New Zealand, showed periods of thermal stratification of the water column, causing a high concentration of phytoplankton in deeper water, at the thermocline. Mussels were grown simultaneously at 5 and 17 m, for up to 96 days. Mussel growth and condition index data showed no significant differences between the two depth treatments, indicating that in this system there may not be substantial mussel productivity benefits from lowering mussel farms to the deep‐water chlorophyll maximum layer.