大口黑鲈hepcidin真核表达载体构建及表达的初步研究

将大口黑鲈(Micropterus salmoides)抗菌肽hepcidin cDNA定向插入真核表达载体pPICZαA,通过SacI酶切线性化重组表达质粒pPICZαA-hepcidin,电转化毕赤酵母P.pastoris GS115,PCR扩增筛选,甲醇诱导表达等进行了hepcidin真核表达工程菌株的构建及诱导表达。结果显示:hepcidin cDNA成功插入表达载体pPICZαA,并整合到酵母基因组中;经甲醇诱导,RT-PCR检测显示hepcidin cDNA在酵母细胞中成功转录。     

牙鲆变态早期cDNA文库的构建和parvalbumin基因的克隆

在前期的研究中,克隆到牙鲆变态早期和变态前差异表达的parvalbumin基因片断cDNA。为了克隆parvalbumin基因的全长cDNA,构建了变态早期(孵化后23d仔鱼)牙鲆头部的全长cDNA文库,并对文库的滴度和插入片段大小进行了评估。同时采用锚定PCR法从cDNA文库筛选出parvalbumin基因的全长cDNA。结果表明,所构建的牙鲆变态早期头部cDNA文库的滴度为1.2×106PFU·mL-1,插入片段的平均长度为1000bp左右。parvalbumin基因的全长cDNA的长度为603bp,编码109个氨基酸,含有两个EF手型钙离子结合域:DQDGSGFIEEEEL(52～64)和DSDGDGKIGVEEF(91～103),以及一个ATP/GTP结合位点模序:ACGLAGKS(33～40),是一种典型的钙离子结合蛋白。通过parvalbumin基因氨基酸序列的同源比较分析表明,牙鲆parvalbumin基因是比较保守的蛋白,与其它鱼类享有61.5%～68.8%序列同源。系统分析表明,牙鲆的parvalbumin基因与狭鳕(Theragrachalcogramma)最为接近。     

翘嘴鳜生长激素cDNA克隆及其真核表达载体的构建

根据GenBank上大眼鳜生长激素cDNA序列(收录号:AY155227)设计了1对特异性引物,应用RT-PCR技术从翘嘴鳜(Siniperca chuatsi)脑垂体中克隆了生长激素基因(scGH)编码区序列并对其真核表达载体的构建进行了研究。结果显示:扩增片段全长615 bp,共编码204个氨基酸。该序列与GenBank上大眼鳜生长激素cDNA序列同源性为99.84%。将scGHcDNA序列定向插入pYES2/CT真核表达载体中,经过筛选、酶切和测序,证明重组子中确实插入了翘嘴鳜GH片段。     

中华绒螯蟹卵巢EJ01基因在大肠杆菌BL21（DE3）中的表达

扩增中华绒螯蟹（Eriocheir sinensis）卵巢EJ01（GenBank检索号：AYl85917）的cDNA全序列得到编码区序列，并将其插入原核表达载体pET28b中，构建成表达质粒pET28b-EJ01。该表达质粒在大肠杆菌（Escherichia coli）BL21（DE3）中经IPTG诱导表达，得到EJ01-HIS6融合蛋白。SDS-PAGE电泳分析表明，该融合蛋白的相对分子质量约为32000。经镍离子柱亲和层析进一步纯化得到纯度较高的EJ01-HIS6融合蛋白。     

中华绒螯蟹蜕皮抑制激素1(Ers-MIH1)-GST融合蛋白在大肠杆菌中的表达

蜕皮抑制激素(molt—inhibiting hormone，MIH)属甲壳动物高血糖激素(crustacean hyperglycemic hormone，CHH)家族。MIH抑制Y-器官蜕皮激素的合成。以中华绒螯蟹(Eriocheir japonica sinensis)眼柄总RNA为模板，根据中华绒螯蟹蜕皮抑制激素1(Eriocheir japonica sinensis molt—inhibiting hormone-1，Ers—MIH1)基因序列设计引物，用RT—PCR的方法获得了Ers—MIH1基因成熟肽的cDNA片段。序列分析表明，Ers—MIH1基因成熟肽编码区含有228bp，编码75个氨基酸残基，与已发表的序列一致。将该cDNA片段插入到中间载体pMD18-T中，酶切后再将该片段插入到pGEX-4T-1表达载体中，构建成表达质粒pGEX-4T—MIH1。在BL21细胞中经IPTG诱导表达，得到GST—MIH1的融合蛋白。SDS—PAGE分析表明，经0．1mmol／L IPTG诱导4h，发现大量GST—MIH1融合蛋白表达，在分子量(Mr)为34kD处有1条特异的蛋白质条带。中华绒螯蟹蜕皮抑制激素1融合蛋白的成功表达为进一步深入研究MIH在中华绒螯蟹蜕皮过程中的作用机制奠定了基础。     

牙鲆SRF基因的克隆及真核表达载体的构建

采用RACE-PCR技术,从牙鲆(Paralichthys olivaceus)组织中克隆了血清应答因子(SRF)基因全长序列,该序列全长2 477 bp,开放阅读框1 503 bp,编码500个氨基酸。通过氨基酸同源序列比对,牙鲆SRF与其它物种的同源性较高,在氨基酸序列的N端具有NLS结构域和高度保守的MADS结构域。用PCR方法扩增SRF基因的编码区片段,克隆到p EGFP-N1载体中,构建真核表达载体p EGFP-N1-SRF,将重组质粒转染牙鲆胚胎细胞,在荧光显微镜下观察转染细胞有绿色荧光蛋白表达。荧光定量PCR和Western blot实验进一步证实,SRF在转染细胞中高表达。说明真核表达载体p EGFP-N1-SRF构建成功,为进一步研究SRF在牙鲆变态发育中的作用奠定了实验基础。     

草鱼Mx蛋白基因的克隆与原核表达

根据已报道的Mx蛋白cDNA序列设计合成特异引物，应用逆转录-聚合酶链式反应(RT-PCR)方法，从草鱼呼肠孤病毒(GCRV)诱导的草鱼(Ctenopharyngodon idellus)肝脏总RNA中扩增获得Mx蛋白cDNA，回收纯化后克隆到pGEM-T Easy Vector系统的T载体上。重组子的序列分析表明：所克隆的Mx蛋白cDNA长722bp，编码240个氨基酸，包括1个三联GTP结合区域和1个发动蛋白(dynamin)族特征的序列。与已知鱼类Mx蛋白基因序列比较表明，草鱼Mx蛋白基因序列与其它鱼类Mx基因相应序列具有较高的同源性，其中与斑马鱼Mx蛋白E型基因碱基序列比较同源性最高，为87．1％。将此基因改造后定向克隆至原核表达质粒pBV220，构建成重组草鱼Mx蛋白基因表达质粒pBVgcMx，并在大肠杆菌中获得高效表达，重组草鱼Mx蛋白的表达量占菌体总蛋白的26．6％。Q-sephrose FF层析柱分离纯化的重组草鱼Mx蛋白纯度达95．6％。     

中华绒螯蟹卵巢EJO1基因在大肠杆菌BL21(DE3)中的表达

扩增中华绒螯蟹(Eriocheir sinensis)卵巢EJO1基因(GenBank检索号:AY185917)的cDNA全序列得到编码区序列,并将其插入原核表达载体pET28b中,构建成表达质粒pET28b-EJO1。该表达质粒在大肠杆菌(Esche-richia coli)BL21(DE3)中经IPTG诱导表达,得到EJO1-H IS6融合蛋白。SDS-PAGE电泳分析表明,该融合蛋白的相对分子质量约为32 000。经镍离子柱亲和层析进一步纯化得到纯度较高的EJO1-H IS6融合蛋白。     

禽多杀性巴氏杆菌PlpB基因交叉保护力研究

根据报道的PM70菌株的PlpB基因序列,设计PlpB基因一对寡核苷酸引物。以C48-1菌株为实验材料,成功扩增PlpB基因的全序列,该片段共831bp。将扩增基因插入原核表达载体pGEX-KG,成功构建了重组表达质粒pKG-PlpB,并对pKG-PlpB重组表达质粒进行序列测定,结果表明:扩增序列与GenBank上所报道的PM70株的PlpB基因序列核苷酸同源性在99%以上。将该重组质粒转化大肠杆菌BL21(DE3)感受态细胞,经诱导获得大小约56KDa的表达蛋白。Western blot检测表明,该表达蛋白具有良好的反应原性。     

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).     

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.     

Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae

The indiscriminate use of antibiotics and chemicals in shrimp hatcheries has led to biomagnification and that in turn could lead to rejection of a whole consignment. The application of the bioencapsulation technique as a tool for curative treatment in shrimp larvae was investigated. Herbs having antibacterial properties such as Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia (methanolic extracts) were bioencapsulated in Artemia and fed to Penaeus monodon post larvae PL 1–25. The post larvae were reared in a medium inoculated with pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Vibrio sp. Post larvae reared in the non-inoculated water and fed with non-enriched Artemia exhibited 90% survival, highest specific growth rate (12.43%) and reduced bacterial load. P. monodon reared in the bacterial inoculated water and fed with the non-enriched Artemia exhibited the lowest survival (10–30%), specific growth rate (8.42–9.1%) and increased bacterial load (2.86 × 10³ to 3.76 × 10⁵ cfu/g). The methanolic extracts of the herbs helped to increase survival and specific growth rate and reduced bacterial load in the P. monodon culture system. Among the three herbal extracts, P. corylifolia enriched Artemia fed post larvae showed the tendency to higher survival (>50%), growth rate (11.5 averaged) and low bacterial load (1.12 × 10⁵ cfu/g). This revised version was published online in August 2006 with corrections to the Cover Date.