剑尾鱼卵黄蛋白原C(Vg C)全长cDNA的克隆及表达

利用逆转录聚合酶链式反应(RT-PCR)和cDNA末端快速扩增(RACE)等方法,克隆获得剑尾鱼卵黄蛋白原C(Vg C)基因的全长cDNA序列。剑尾鱼Vg C基因cDNA序列全长4 011 bp,其5′非编码区包含12 bp和3′非编码区包含246 bp;含有一个3 753 bp的开放阅读框(ORF),编码1 250个氨基酸,推测其编码氨基酸分子量大小为141.7 ku,编码氨基酸序列与其他鱼类卵黄白原C编码氨基酸序列相似性在44%～85%。荧光定量PCR结果显示,Vg C在剑尾鱼肝脏中表达量最高,脾、肾、卵巢中有微量表达,脑、肌肉、鳃中几乎没有检测到表达;对不同时间暴露在雌激素中剑尾鱼肝脏进行实时荧光定量PCR表达分析的结果表明,Vg C在剑尾鱼肝脏中第5天表达量最高,随后降低,第9天后维持相对较低的表达量。研究首次克隆了剑尾鱼Vg C基因全长cDNA序列,并对Vg C在剑尾鱼体内表达组织器官分布及雌激素诱导后不同时间表达谱进行了初步研究,为剑尾鱼生殖生理及环境污染物监测应用等不同领域研究打下重要基础。     

卵黄蛋白原在剑尾鱼体内不同组织的分布及雌激素作用对其表达的影响

为研究卵黄蛋白原(vitellogenin,vg)在剑尾鱼(Xiphophorus helleri)不同组织器官的表达情况,以及了解雌激素对不同组织中Vg表达的诱导作用,将成年雌、雄剑尾鱼及幼鱼暴露于50 μg/L的17β-雌二醇(E2)溶液中20 d,未暴露的剑尾鱼作对照,采用免疫组化法对各处理组剑尾鱼进行Vg的组织定位研究.结果表明,剑尾鱼Vg主要分布于成熟雌鱼的肝脏、脾脏和肠的血管、淋巴管,在正常幼鱼及雄鱼的以上各组织中均不表达.17β-雌二醇暴露下,剑尾鱼雌、雄成鱼和幼鱼的肝脏以及肾、脾、肠等组织的血管及淋巴管均呈Vg阳性.但Vg颗粒只在肝实质细胞中观察到,在其他组织中如脾脏、肠固有结缔组织等,Vg颗粒仅出现于血管,在细胞内未观察到.以上结果表明,剑尾鱼Vg在肝细胞中合成,通过血液循环到达其他组织;雌激素可诱导雌、雄剑尾鱼及幼鱼肝组织中Vg的表达.剑尾鱼幼鱼及雄鱼Vg的非正常表达可应用于环境雌激素效应评价.     

瓦氏黄颡鱼血清卵黄蛋白原、钙离子的变化与性腺发育的关系

在雌性瓦氏黄颡鱼性腺发育过程中,随着卵巢中卵黄蛋白的积累,性腺系数的增加,血清中钙离子浓度升高,同时血清中的卵黄蛋白原含量也相应地增加。然而在雄鱼的性腺发育过程中血清钙离子无明显变化。产卵后,雌鱼卵黄蛋白原、钙离子含量降至最低水平。     

热应激对剑尾鱼HSP70家族两成员基因表达的影响

采用RT-PCR方法扩增实验动物剑尾鱼（Xiphopohorus helleri）纯系RR-B的HSP70家族两个成员的eDNA片段，并将其克隆到PMD18-T载体中测序，将测序结果与GenBank中的核苷酸序列和推导的氨基酸序列进行同源性比较。同时利用RT-PCR半定量方法研究热应激时两个成员在剑尾鱼组织中的表达。通过克隆获得了剑尾鱼的HSP70家族两个新成员的cDNA片段，两序列均包含HSP70家族的特征性签名序列和热休克蛋白的定位序列；通过对克隆片段与已发表的青锵（Oryzias latipes）等鱼类HSP70的核苷酸序列和其编码的氨基酸序列同源性比较，发现核苷酸序列同源性较高，成员一和成员二分别为85％-89％和82％-99％；氨基酸序列同源性更高，成员一和成员二分别为97％-99％和93％-99％。一般条件下，两成员在剑尾鱼肝脏、脾脏、肾脏和心脏中不表达，但热应激能刺激成员一在剑尾鱼脾脏中表达，成员二在肝脏、脾脏、肾脏和心脏中强烈表达，并可能在参与热应激保护方面起更大作用。     

剑尾鱼卵黄脂磷蛋白的纯化及免疫分析

采用Sephacryl S-300凝胶过滤层析柱和HiTrap Q阴离子交换柱从卵黄生成期的雌性剑尾鱼(Xiphophorus helleri)卵巢组织提纯了卵黄脂磷蛋白(Lv)。已确定被纯化的剑尾鱼Lv在Native-PAGE(4%~7.5%)电泳中分子量为530 kD左右。Native-PAGE(4%~7.5%)电泳后的凝胶分别进行糖蛋白染色、脂蛋白染色及磷蛋白染色。结果表明,剑尾鱼Lv是一种富含糖、脂、磷的蛋白。用纯化的剑尾鱼Lv免疫大白鼠获得鼠源多克隆抗血清。用免疫双扩散的方法测定Lv抗血清的效价为l∶32。Western-blotting检测显示抗血清的特异性较好;卵黄蛋白原(Vtg)和Lv抗血清之间存在明显的免疫交叉反应,表明Vtg和Lv两者具有相同的免疫原性。免疫双扩散检测表明,抗Lv血清表现出明显的雌性特异性和种的特异性。     

剑尾鱼 2 种卵黄蛋白原全长 cDNA 的克隆及序列分析

卵黄蛋白原是环境雌激素效应研究的重要生物标志物,广泛应用于水环境内分泌干扰物污染的评价。本研究利用RT-PCR和cDNA末端快速扩增法(RACE)克隆获得了剑尾鱼(Xiphophorus helleri)的2种卵黄蛋白原基因—VgA和VgB,并对其基因结构和系统进化进行分析。VgA基因cDNA序列全长5159bp,开放阅读框(ORF)5058bp,编码1685个氨基酸。VgB基因cDNA序列全长5349bp,开放阅读框(ORF)5067bp,编码1688个氨基酸。2种cDNA序列均具有与已发现的卵黄蛋白原分子相似的主要特征和功能域,包括Vitellogenin结构域、VWFD结构域和多聚丝氨酸区域,且与已知其他鱼类卵黄蛋白原基因有较高的同源性。     

瓦氏黄颡鱼卵黄脂磷蛋白(Lv)的纯化、性质鉴定及其抗血清的研制

采用凝胶过滤和离子交换两种层析技术,从瓦氏黄颡鱼Ⅳ期卵巢粗提液中分离、纯化出卵黄脂磷蛋白(Lv),采用糖、磷、脂蛋白染色技术验证分离、纯化的蛋白为Lv,该Lv在非变性条件下分子量约为230ku,在SDS变性条件下分子量约为106ku。纯化的瓦氏黄颡鱼Lv经检测显示含有类胡萝卜素,但没有二硫键,对热相对稳定。利用纯化的瓦氏黄颡鱼Lv,制备了兔抗瓦氏黄颡鱼Lv多克隆抗血清。通过双向免疫扩散法测得Lv抗血清的效价为1∶32,还发现瓦氏黄颡鱼卵黄蛋白原(Vtg)和Lv之间有明显的免疫交叉反应性,说明两者具有相同的免疫原性;Western-blotting检测显示抗血清的特异性较好,并能特异性地识别Vtg。     

野生与养殖黄颡鱼鱼卵的营养成分比较

以贵州省铜仁锦江河漾头河段野生黄颡鱼鱼卵和同一区域内养殖黄颡鱼鱼卵为研究对象,采用常规生化方法测定了野生与养殖黄颡鱼鱼卵氨基酸组成和含量,采用气相色谱技术检测了野生与养殖黄颡鱼鱼卵脂肪酸组成和含量。氨基酸测定结果表明,野生黄颡鱼鱼卵的氨基酸总量、必需氨基酸总量、鲜味氨基酸总量显著低于养殖黄颡鱼鱼卵(P0.05),第一限制性氨基酸均为甲硫氨酸+半胱氨酸,必需氨基酸指数分别为55.60和59.12。脂肪酸测定结果表明,野生黄颡鱼鱼卵的饱和脂肪酸总量极显著高于养殖黄颡鱼鱼卵(P0.01),单不饱和脂肪酸总量二者差异不显著(P0.05),多不饱和脂肪酸总量极显著低于养殖黄颡鱼鱼卵(P0.01),但野生黄颡鱼鱼卵的二十碳五烯酸和二十二碳六烯酸的总量极显著高于养殖黄颡鱼鱼卵(P0.01),野生黄颡鱼鱼卵的n-3系列的多不饱和脂肪酸总量与n-6系列不饱和脂肪酸总量的比值(1.57)低于养殖黄颡鱼鱼卵(1.80)。     

大阪鲫鱼血清雌性特异蛋白的鉴定

鱼类是在卵母细胞外合成卵黄的动物，具体说是在肝脏中合成的，经过血液循环输送到卵母细胞，首先在肝脏中合成的是卵黄蛋白的前体——卵黄蛋白原(Vitellogenin)，卵黄蛋白原在雌激素作用下，雌体血清中就会出现一种雌性特异蛋白成分，K．Aida(1973)称此蛋白为血清雌性特异蛋白。近年来，作者在鱼类血液分析方面的工作证明，雌雄鱼血清存在差异是普遍的，并利用醋酸纤维薄膜电泳对雌性特异蛋白化学特性进行了鉴定。     

鲤春病毒血症病毒糖蛋白的截短表达及其免疫原性分析

为了揭示鲤春病毒血症病毒(spring viremia of carp virus,SVCV)糖蛋白编码基因的主要免疫原性决定区域,本研究对SVCV的糖蛋白基因进行截短表达,并用纯化的重组蛋白作为免疫原制备了兔抗血清以分析其免疫原性。通过SOSUI以及DNAStar 6.0软件对SVCV糖蛋白基因的跨膜区、亲水性以及抗原表位进行分析后,采用RT-PCR对该基因主要抗原决定区域编码片段进行扩增,并构建重组表达质粒p GEX-Gtr,对其进行诱导表达后获得截短的SVCV糖蛋白的重组蛋白。将表达的重组蛋白进行纯化复性后,作为免疫原制备兔抗血清,采用ELISA法检测其效价,采用免疫印迹以及间接免疫荧光技术检测该重组蛋白的免疫原性。研究结果显示,截短表达的糖蛋白编码基因长1317 bp,编码439个氨基酸(29~467),推测分子量为49.6 k D。利用该重组蛋白制备的兔抗血清,其与重组蛋白的反应效价为1∶64000,。免疫印迹及间接免疫荧光结果显示,该兔抗血清与SVCV-HN株能发生特异性反应,表明该重组蛋白与天然的病毒表面糖蛋白的免疫原性无差异。上述研究结果表明,截短表达的重组蛋白具有很好的免疫原性,可应用于免疫诊断技术研发与基因工程疫苗的研制。     

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 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).     

Evaluation of a Fast Method Based on the Presence of Two Restriction Sites in the Mitochondrial ND5 (mt ND5) Gene for the Identification of Scomber Species

The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus.     

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.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

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.