4株长牡蛎(Crassostrea gigas)致病性哈维氏弧菌(Vibrio harveyi)鉴定及其毒力基因检测

哈维弧菌(Vibrio harveyi)是海水鱼养殖中一种重要的病原菌。该研究根据已知rbsB (核糖体结合蛋白)基因序列设计引物,扩增得到哈维弧菌354 (V.harveyi 354) rbsB基因的全序列(GenBank序列号MF797015),预测该序列可编码292个氨基酸,分子量为30.7 kD,理论等电点为5.05,亲水性系数为0.043,为疏水性蛋白。根据RbsB氨基酸构建的系统进化树可以发现哈维弧菌RbsB蛋白和欧文弧菌(V.owensii CAIM 1854)的关系最近。构建了pGEX-4t-1-rbsB重组质粒并转化至大肠埃希菌BL21 (DE3)中,得到的重组蛋白的相对分子量约59 kD,在37 ℃、IPTG浓度为0.6 mmol·L^–1诱导8 h时表达量最高。     

大菱鲆致病性溶藻弧菌SR1的外膜蛋白及其抗原性分析

用十二烷基肌氨酸钠(Sarkosyl)抽提结合超速离心的方法提取了一株大菱鲆致病性溶藻弧菌(Vibrio alginolyticus)SR1和其他7株弧菌的外膜蛋白。通过SDS-PAGE图谱分析比较了这8株弧菌外膜蛋白的组成,结果表明,8株弧菌的外膜蛋白电泳一般可得到6-12条条带,其分子量多集中在65-106 kD和28-48 kD,其中36 kD的蛋白带为8株弧菌所共有。用兔抗SR1全菌血清进行Western-blot印迹显示,菌株SR1的外膜蛋白条带中有6条发生了阳性反应,其分子量分别为73 kD、48 kD4、5 kD3、9 kD、36 kD和32 kD。而其他7株弧菌的外膜蛋白与兔抗SR1血清也发生程度不等的阳性反应,这些阳性反应条带的分子量集中在65-73 kD、45-48 kD和36-41 kD之间,其中36 kD的外膜蛋白在8株弧菌中均出现明显的阳性反应,说明36 kD的外膜蛋白是这8株弧菌共有的特异性抗原。     

鸢乌贼酶解产物的抗氧化稳定性与功能特性

以葡聚糖、褐藻寡糖、葡萄糖、乳糖为糖基供体实现对鸢乌贼(Symplectoteuthis oualaniensis)肌原纤维蛋白糖基化改性,分析了不同糖的种类及改性时间对蛋白溶解性、结构、乳化性、起泡性等功能性质的影响。结果表明,肌原纤维蛋白与不同种类的糖质量比1∶1反应12 h后,SDS-PAGE分析显示糖基化产物分子量明显增大,小分子糖与肌原纤维蛋白反应更快;糖基化反应使肌原纤维蛋白的溶解性显著提高,表面疏水性降低;此外,肌原纤维蛋白二级结构明显改变,β-转角含量显著升高,β-折叠含量降低。糖基化反应未能改善肌原纤维蛋白的热稳定性。反应后的肌原纤维蛋白具有更好的起泡性及起泡稳定性,但乳化性和乳化稳定性降低。肌原纤维蛋白糖基化后在高离子浓度氯化钠(NaCl)溶液中比在低离子浓度溶液中具有更高的乳化和起泡能力。同时,经糖基化改性的蛋白在低离子浓度时的溶解性和起泡能力也能得到明显改善。     

Characterization of β-N-Acetylhexosaminidase from rhizostomous jellyfish, Rhopilema asamushi, Mesogloea

β-N-Acetylhexosaminidase (EC 3.2.1.52) was purified from rhizostomous jellyfish mesogloea and characterized. Using two purification steps, this enzyme was purified up to 27.4-fold with a recovery rate of 46% compared with crude extract. The molecular weight of the enzyme was estimated to be about 136 kDa, composed of subunit molecular weights of 68 kDa. The enzyme activity was inhibited by SH-reagents, indicating that it contains a SH-group in its active site. The enzyme has a high affinity for pNPGlcNAc with Km value of 0.021 mM. The rate of hydrolysis of N-acetylchito-oligosaccharides tended to decrease with increasing degree of polymerization of the substrate. The parameters of k _cat were 92.0 s⁻¹ for pNPGlcNAc, 38.2 s⁻¹ for GlcNAc₂, 14.0 s⁻¹ for GlcNAc₃, 4.1 s⁻¹ for GlcNAc₄, 1.6 s⁻¹ for GlcNAc₅, 0.9 s⁻¹ for GlcNAc₆, respectively. These results suggest that this β-N-acetylhexosaminidase is an exo-fashion hydrolytic enzyme involved in chitin degradation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cloning, expression, and characterization of a novel anti-HIV lectin from the cultured cyanobacterium, Oscillatoria agardhii

OAA, the potent anti-HIV protein from Oscillatoria agardhii NIES-204 belongs to a new lectin family, shows strict binding specificity for high-mannose N-glycans, and has an extremely high association constant in the picomolar range for recombinant gp120, an envelope protein of HIV. In this study we have cloned the gene encoding OAA from the genomic DNA of the cyanobacterium, and efficiently expressed the recombinant lectin (rOAA) in Escherichia coli. The rOAA expressed as a His-tagged fusion protein was recovered in a soluble form and purified in high yield (48 mg/1 l-culture) by metal chelate chromatography. The fusion protein was cleaved with factor Xa, and the resulting rOAA was isolated in a final yield of 14.8 mg/1 l-culture by reversed-phase HPLC. Both the N-terminal sequence and the molecular mass of rOAA were found to be identical with those of OAA. The rOAA was fully functional with the same properties as OAA, as evidenced by hemagglutination activity, hapten-inhibition test, and binding specificity for high-mannose-type N-glycans. This rOAA should be applicable as a specific probe for high-mannose N-glycans and should contribute to elucidation of the molecular basis of its strict carbohydrate-binding specificity and potent anti-HIV activity.     

急性低盐胁迫下红鳍东方鲀幼鱼IgM、NKCC1和Hsp70基因的表达

为探讨红鳍东方鲀(Takifugu rubripes)耐低盐的分子机制,以自然海水组为对照,利用实时荧光定量PCR(q RT-PCR)技术,分析红鳍东方鲀幼鱼在盐度16、12、8和4胁迫下,鳃和肾中免疫球蛋白M(immunoglobulin M,IgM)、钠钾氯协同转运蛋白1(Na-K-Cl cotransporter 1,NKCC1)和热休克蛋白70(heat shock protein 70,Hsp70)3个基因的表达情况。结果表明,3个基因在鳃和肾中均有表达,IgM和Hsp70基因在鳃和肾中的表达量均无显著性差异(P0.05),而NKCC1基因在鳃中的表达量显著高于肾(P0.05)。同一盐度下,随着时间的增加,鳃中IgM基因的表达量大致呈现先降低后升高的趋势,肾中则呈现先降低后升高再趋于平稳的趋势;同一时间内,鳃中低盐度组与对照组的IgM基因表达量差异比肾中差异更为明显。在鳃中,相同时间内NKCC1基因在各低盐度组的表达量低于对照组,尤其在6h和72h两个时间点时显著低于对照组(P0.05);肾中各时间点的表达量基本都高于0h表达量。在相同时间内,3h、6h、24h、72h的各低盐度组,在鳃中,Hsp70基因的表达量均与同一时间对照组之间差异明显(P0.05);在肾中,从6h开始,各低盐组与对照组之间差异性显著(P0.05)。以上结果表明,红鳍东方鲀幼鱼IgM、NKCC1、Hsp70 3个基因的表达量在不同盐度不同时间下存在差异,由此推测这3个基因对红鳍东方鲀幼鱼的渗透压调节起重要作用。     

Protein Hydrolysates and Ultrafiltration Fractions Obtained from Krill (Euphausia superba): Nutritional,Functional, Antioxidant,and ACE-Inhibitory Characterization

ABSTRACT

Krill (Euphausia superba) was hydrolyzed by proteolytic enzymes in order to produce multifunctional bioactive peptides, and their functional properties were evaluated. Krill protein hydrolysate (KPH) by pepsin with 4-h hydrolysis showed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging and angiotensin I converting enzyme (ACE) inhibitory activities. The solubility and foaming properties of KPH were higher than those of the unhydrolyzed krill protein at a wide range of pHs. KPH was further fractionated based on molecular weight. The 1- to 3-kDa peptide fraction exhibited the highest DPPH scavenging activity (IC₅₀ value of 0.5 mg/mL), oxygen radical absorbance capacity (497.39 ± 4.31 µM TE/mg fraction), 2,2-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid cation radical scavenging activity (48.41 ± 0.23 µM TE/mg fraction), and reducing power (110.40 ± 2.07 µM TE/mg fraction). However, the < 1-kDa peptide fraction exhibited a higher ACE inhibitory activity than that of other fractions. The 1- to 3- and < 1-kDa peptide fractions are rich in aromatic and hydrophobic amino acids, respectively.     

Influence of Different Hydrolysis Processes by Trypsin on the Physicochemical,Antioxidant, and Functional Properties of Collagen Hydrolysates from Sphyrna lewini,Dasyatis akjei,and Raja porosa

ABSTRACT

Acid soluble collagen hydrolysates from the cartilages of Sphyrna lewini, Dasyatis akjei, and Raja porosa on different hydrolysis conditions by trypsin were prepared and named as S, D, and R, respectively. The hydrolysates (S1, D1, and R1) obtained from hydrolysis in pH 2.5, 3 h, 37°C presented wonderful foaming and emulsifying capacities, caused by their high average molecular weights (AMWs), high degree of Pro and Lys hydroxylation, and imino acid contents (17.1, 15.3, and 14.1%). At the concentration of 0.1% (w/v), the foaming capacities of S1, D1, and R1 were 104.75 ± 2.57, 63.87 ± 2.73, and 76.87 ± 2.02%; and the emulsifying activity indexes of them were 116.07 ± 1.89, 91.04 ± 1.79, and 123.85 ± 2.14 m²/g, respectively. Conversely, hydrolysates (S2, D2, and R2) obtained from hydrolysis in pH 7.8, 3 min, 37°C exhibited strong scavenging activity on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (EC₅₀ 0.568, 0.680, and 1.634 mg/mL), hydroxyl radical (EC₅₀ 0.253, 0.376, and 0.438 mg/mL), and moderate reducing power (0.0465–0.4702 at the concentration of 5 mg/mL), due to their low AMWs. The results indicated that S1, D1, and R1 could be used as emulsifiers and foaming agents, and S2, D2, and R2 could be used as natural antioxidants in food systems.     

Antibody specificities in Atlantic salmon, Salmo salar L., against the fish pathogens Vibrio salmonicida and Vibrio anguillarum

Abstract. Specificities of polyclonal salmon antisera made against the fish pathogens Vibrio salmonicida and Vibrio anguillarum were studied. Using ELISA and Western blot techniques, antisera made against V. salmonicida or V. anguillarum serovar 1 demonstrated high responses against the homologous bacterium or its isolated LPS. In contrast, antisera obtained after immunization with V. anguillarum serovar 2 displayed low antibody titres against homologous antigens. Elcctrophoretic transfer of SDS-PAGE separated V. salmonicida LPS antigen to nitrocellulose strips and subsequent immunostaining with salmon antisera revealed a strong reaction exclusively in the low molecular weight region (<14kD). On the other hand, immunoblots of V. anguillarum LPS preparations using salmon immunesera raised against this species showed a heterogenous staining pattern ranging from high to medium LPS-size. In addition, most of the salmon antisera made against V. anguillarum serovar 2 also reacted with a low molecular weight LPS antigen band.     

Euclinostomum heterostomum and E. ardeolae in tilapia species of Aswan Governorate,Egypt: morphological,molecular, and histopathological characterization

Euclinostomum heterostomum and Euclinostomum ardeolae, both encysted metacercariae (EMC), were found to infect farmed Oreochromis niloticus in the Sahary fish hatchery and wild O. niloticus, Sarotherodon galilaeus, and Tilapia zillii in Lake Nasser, Aswan Governorate, Egypt, at a prevalence of 25.25% and an infection intensity of 1–14 EMC/fish. Macroscopic and microscopic examinations were used to identify them morphologically. PCR amplification, sequencing of the 28S large ribosomal RNA region, and phylogenetic analysis were used for molecular characterization. E. heterostomum and E. ardeolae were isolated from farmed O. niloticus kidneys, peritoneum, abdominal cavity, and under the gills attached to the head bone at prevalence of 13.5% and 6%, respectively, and from wild O. niloticus, S. galilaeus, and T. zillii at a prevalence of 15% and 4.5%, 18% and 10%, and 22.5% and 11.5% respectively. The molecular analyses of rRNA-28S marker revealed that the two Euclinostomum species examined are related yet distinct. This is the first molecular and phylogenetic evidence linking E. ardeolae to Euclinostomum. The present study added two new sequences to the GenBank databases for the genus Eulinostomum from Egypt, with accession numbers MW604803 and MW604806. Euclinostomum spp. have been shown to have detrimental and destructive effects on the kidneys of infected fish; these effects may eventually result in the parasites’ cysts replacing the kidney’s tissues. The kidneys of tilapia spp. infected with Euclinostomum spp. displayed degenerative alterations, with dilated renal tubules associated with migratory tunnels composed primarily of leucocytic infiltrations. Euclinostomum spp. exhibited clear hemophagic characteristics.

     

中华绒螯蟹代谢蛋白互作网络构建及分子功能、亚细胞定位和途径分析

为了构建中华绒螯蟹代谢过程研究的系统工具，实验在已经构建的中华绒螯蟹蛋白互作网络的基础上，首先采用邻接节点注释法对未知蛋白的分子功能进行预测。随后采用GO回溯法，构建了代谢蛋白网络并对网络中蛋白分子功能、亚细胞定位和途径分布进行了分析。分子功能注释中，确定了932个蛋白的分子功能，占所有未知分子功能蛋白的97%。最终构建的代谢蛋白互作网络包含2 045个蛋白及这些蛋白之间的15 927条互作关系。网络中94.2%(1 926/2 045)的蛋白具有亚细胞定位信息，大多分布于有膜细胞器中；96.1%的蛋白(1 966/2 045)具有分子功能信息，大多具有催化活性和结合活性。进一步对确定了分子功能和亚细胞定位的蛋白在40个KEGG子系统中的分布进行分析，发现参与翻译和氨基酸代谢过程的蛋白较多，也有一部分参与免疫和运输过程。本实验结果可为中华绒螯蟹代谢相关的蛋白功能、定位的研究提供重要的数据参考，对系统研究中华绒螯蟹代谢过程及代谢相关疾病具有重要价值。     

First detection of koi herpesvirus from koi,Cyprinus carpio L. experiencing mass mortalities in Iran: clinical,histopathological and molecular study

Koi herpesvirus (KHV) is the aetiological agent of an emerging disease (KHVD) associated with mass mortalities in koi and common carp and reported from at least 30 countries. We report the first detection of KHV from koi in Iran using clinical, histopathological and molecular studies. KHV‐infected fish showed reduced swimming activity, sunken eyes and increased mucus production on skin and fins. On post‐mortem examination, gill necrosis was observed in the majority of fish. Histopathologically, the gill showed diffuse necrosis of the branchial epithelial cells. Margination of chromatin was detected in gills, kidney, heart, spleen, intestine and brain. In addition, sequence analyses of the TK gene, ORF 136 and marker I and II, demonstrates that Iranian KHV isolates were identical and classified as variant A1 of TUSMT1 (J strain) and displayed the I⁺⁺II⁺ allele of this Asian genotype.     

The effects of starving and feeding on Dover sole (Solea solea,Soleidae, Linnaeus, 1758) stress response and early larval development

In the view of an urgent necessity to improve the quantity and the quality of farmed fish species, there is a strong need to improve our basic knowledge on the effects of first feeding during the developmental stages of fish larvae. High mortality, mainly due to food deprivation or inappropriate food quality, has been observed in many larval fish species, but knowledge about the morphological, biochemical and molecular processes related to this topic is still poorly understood. The understanding of the early larval ontogeny as well as the larval nutritional requirements and the molecular and cellular mechanisms elicited by fish larvae during food deprivation and starvation are thus of primary importance. At this regard, this study investigates, in Dover sole larvae, the effects of starvation and starving/re‐feeding procedures at a morphological, histological, biochemical and molecular level. The results evidenced that starved larvae progressively decrease in growth, lipid content, affected their gastrointestinal tract and muscle development and increased cortisol and heat shock protein 70 levels. On the contrary, starved and re‐fed larvae showed, after the restoration of a favourable feeding condition, a compensatory growth. In conclusion, this is the first study analysing through a multidisciplinary approach the effects of food deprivation on the development of an important economic species, the Dover sole.     

Isolation of a novel hepatic CYP2-related protein from channel catfish, Ictalurus punctatus

Channel catfish (Ictalurus punctatus) have been previously shown to express two major cytochrome P450 (CYP) protein bands that are cross-reactive with anti-CYP2K1 (rainbow trout, Oncorhynchus mykiss) antibodies on Western blots. These proteins appear to be the major constitutive CYPs in channel catfish and show distinct sex- and age-specific variations in expression. Because I. punctatus is an important agricultural and ecological commodity, and because it displays a high degree of resistance to the toxic effects of many pesticides, the molecular and catalytic characteristics of its biotransformation systems are of interest to those in areas of environmental science and aquaculture research. Using a chromatographic method similar to that employed in the purification of other fish CYP2 enzymes, a single CYP2-related protein (CM-HA3) was isolated from channel catfish hepatic microsomes. The isolated protein displays a relative molecular mass of approximately 47 kDa, and a CO-reduced difference spectrum _max of 449.6 nm. The sequence of 15 residues at the amino-terminal of CM-HA3 is 27% identical to both CYP2K1 and CYP2M1 isoforms of rainbow trout. Correlational analysis was employed to characterize potential substrates for this isoform, but no significant relationship was observed with E₂ hydroxylation, testosterone hydroxylation, or 7-ethoxycoumarin O-deethylase activities. These data indicate that CM-HA3 is a CYP2 family protein, with as yet uncharacterized substrate specificities.     

琼胶寡糖的ESI－MS分析研究

首次对6种琼胶寡糖的分子量，采用电喷雾电离质谱（ESI－MS）进行分析测定。结果表明，琼胶寡糖的ESI－MS谱图非常清晰，能准确判断出琼胶寡糖的分子量。这些寡糖只出现[M Na]^ 加成离子，而不出现[M 1]^ 的准分子离子，且[M Na]^ 分子离子峰均特强；同时还出现一系列[M 2Na]^ 的加成离子。当化合物分子量大于1000时，则给出双电荷离子；当化合物分子量小于1000时，则ESI－MS谱出现[2M Na]^ 的系列离子。实验结果揭示，所得6种琼胶寡糖的分子量分别为324、630、936、1242、1548和1854。     

尼罗罗非鱼卵黄脂磷蛋白的分离纯化与性质鉴定

采用Sephacryl S-300过滤层析和DEAE-Sepharose Fast Flow离子交换层析相结合的方法从尼罗罗非鱼(Oreochromis niloticus)成熟卵子匀浆液中分离纯化出了一种高分子量的蛋白。该蛋白能被Schiff试剂、甲基绿和苏丹黑B着色,Western blot显示能被金鱼卵黄脂磷蛋白(lipovitellin,Lv)多克隆抗血清特异性识别,在非变性条件下分子量约为560 k D,在SDS变性条件下分子量约为112 k D,结果表明分离纯化的蛋白是一种含有糖、磷、脂基团的蛋白,符合鱼类Lv的性质,且与金鱼Lv有免疫交叉反应,从蛋白的性质和免疫原性以及分子量大小等角度判断,本研究获得的高纯度蛋白为尼罗罗非鱼卵黄脂磷蛋白;纯化的罗非鱼Lv在反复冻融、37℃及60℃处理条件下均未出现降解,表明罗非鱼Lv比鱼类卵黄原蛋白(Vitellogenin,Vtg)更为稳定。研究结果为罗非鱼Lv抗体的制备奠定了基础。     

Isolation,Purification, and Biochemical Characterization of Trypsin from Indian Mackerel (Rastralliger kanagurta)

Trypsin from viscera of Indian mackerel (Rastralliger kanagurta) was purified by ammonium sulphate precipitation and chromatographic techniques such as size exclusion, ion exchange, and affinity chromatography, with a 14.4-fold increase in specific activity and 18.7% recovery. The molecular weight of the trypsin was estimated to be approximately 26 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified trypsin showed amidase-specific activity which was determined using benzoyl-dl-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for isolated trypsin activity were 9.0 and 50°C, respectively. The purified trypsin was strongly inhibited by soybean trypsin inhibitor (SBTI) and N-p-tosyl-1-lysine chloromethyl ketone (TLCK). Purified trypsin showed almost 40% recovery at high NaCl concentration (30%). The N-terminal amino acid sequence of the first 10 amino acids of purified trypsin was IVGGYESQPH. The Michaelis-Menten constant (K_m) and catalytic constant (K_cat) of purified trypsin were 0.430 mM and 0.77 s^?1, respectively, determined using BAPNA as a substrate. Purified trypsin showed digestion of casein similar to bovine trypsin by the fluorometric method.     

Common pearl oysters in China, Japan, and Australia are conspecific: evidence from ITS sequences and AFLP

ABSTRACT:   To elucidate the species status of Pinctada fucata in China, P. fucata martensii in Japan and P. imbricata in Australia, one population of each taxon was studied using internal transcribed spacer 1 and 2 (ITS1, and ITS2) and amplified fragment length polymorphism (AFLP) markers. ITS1 and ITS2 were 401–405 and 229–237 bp long, respectively. Twenty-nine ITS1 and 15 ITS2 unique genotypes were obtained from 44 and 34 individuals, respectively, with some genotypes shared by two or three populations. In AFLP analysis, each individual exhibited a distinct phenotype. No population had diagnostic markers. Mean genetic divergences within and among the three populations were very low and overlapped (between-population: 0.7–0.9% for ITS1, 0.9–1.3% for ITS2, and 53.3–55.6% for AFLP; within-population: 0.5–0.9% for ITS1, 0.8–1.2% for ITS2, and 50.4–53.6% for AFLP). Low levels of genetic differentiation were observed among the three populations while the Australian population is partially genetically isolated. Under an infinite allele model, genetic differentiation among populations was not significant based on a permutation test. Under an infinite site model, most F _ST values were not significant for ITS data although they were significant for AFLP data. Network analysis using ITS data indicated that individuals from the same population did not cluster together. Analysis of molecular variance ( amova ) demonstrated that > 94% variation was contributed by within-population variation. These findings suggest that the three taxa are conspecific and Pinctada fucata is the correct name.     

Estimation of genetic variation in green tiger prawn,Penaeus semisulcatus by using random amplified polymorphic DNA,inter simple sequence repeat and simple sequence repeat markers

In this study, three molecular markers including random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and simple sequence repeat (SSR) were used to evaluate genetic variation of green tiger prawn Penaeus semisulcatus collected from two geographically isolated environments; located in the Manifa, Arabian Gulf, Saudi Arabia and Ataka, Suez Gulf, Egypt. Genetic parameters included the percentage of polymorphism (P%), effective alleles (Ne), Nei genetic diversity (H) and Shannon index (I), which were calculated based on molecular data. All three marker systems distinguished genetic variation of P. semisulcatus in various levels. The highest polymorphism (91.30%) was obtained with SSR, followed by ISSR (82.26%) and RAPD markers (62.04%), respectively. Our results indicate that SSR appeared to be the best suited molecular assay for assessing the genetic variation between genotypes of P. semisulcatus. The present study indicated that Manifa and Ataka genotypes were closely related. Moreover, the analysis of variability could require more than one DNA‐based molecular marker techniques.