锦鲤疱疹病毒CJ株ORF27基因克隆及生物信息学分析

为了解锦鲤疱疹病毒中国吉林株（KHV-CJ）ORF27基因编码蛋白的结构特征和进化关系,采用镜鲤尾鳍原代细胞增殖KHV-CJ,提取其DNA,经PCR扩增,获得ORF27基因,将其克隆在pMD18-T载体中,构建重组质粒。应用生物信息学方法初步分析KHV-CJ ORF27基因的结构和功能,并与GenBank上公布的三株KHV构建系统进化树。结果显示：获得207bp的ORF27基因,编码69个氨基酸;预测ORF27编码蛋白的理论分子质量为7366.62Da,等电点为4.487;抗原表位预测显示抗原性良好;编码蛋白存在1个N-糖基化位点、4个O糖基化位点和5个磷酸化位点;系统进化树分析表明与锦鲤疱疹病毒美国株（KHV-U）属同一分支。     

锦鲤疱疹病毒-CJ株ORF81基因的克隆及生物信息学分析

为了解锦鲤疱疹病毒中国吉林株(KHV-CJ) ORF81蛋白的结构特征和进化关系,用框镜鲤尾鳍原代细胞增殖KHV-CJ,提取其DNA,经PCR扩增,获得ORF81基因,将其克隆到pMD18-T载体中,构建重组质粒.应用生物信息学方法初步分析KHV-CJ ORF81基因的结构和功能,并与GenBank上已公布的3株KHV构建系统进化树.结果显示,获得了长771 bp的ORF81基因,编码256个氨基酸;预测ORF81基因的理论分子量为28 246.50 u,等电点为8.404;疏水性大于亲水性;信号肽切割部位最可能位于29位的S(丝氨酸);有4个跨膜区;抗原表位预测显示抗原性良好;结构预测显示,不存在N-糖基化位点、存在6个O-糖基化位点和11个磷酸化位点;系统进化树分析显示,与锦鲤疱疹病毒以色列株属同一分支.     

锦鲤疱疹病毒3型ORF126基因克隆及生物信息学分析

为研究锦鲤疱疹病毒3型(KHV-3)ORF126基因编码蛋白的功能,我们对该蛋白的结构特征进行研究分析。本实验采用PCR扩增技术获得KHV ORF126基因的完整序列,构建pMD19-T-ORF 126重组质粒,应用生物信息学方法分析ORF126基因编码蛋白的理化特征。结果显示:KHV ORF126基因翻译合成273个氨基酸;ExPasy预测该蛋白的理论分子量为29.9kDa,等电点为5.11;信号肽的切割部位最可能位于第19~20位氨基酸之间;跨膜区位于第145~168位氨基酸;抗原表位预测显示抗原性良好;编码蛋白不含N-糖基化位点,含有4个O-糖基化位点和17个磷酸化位点。     

传染性造血器官坏死病毒-Sn株基质蛋白基因克隆及生物信息学分析

基质蛋白(matrix protein,M)是传染性造血器官坏死病毒(infectious haematopoietic necrosis virus,IHNV)主要结构蛋白之一,是病毒感染后造成细胞凋亡的主要作用蛋白。为分析IHNV M蛋白的序列及结构特征,研究利用敏感细胞(EPC)培养传染性造血器官坏死病毒-Sn分离株(IHNV-Sn),根据M蛋白基因开放阅读框(open reading frame,ORF)的序列设计引物,利用RT-PCR的方法克隆得到M蛋白全长ORF,并且构建至表达载体pET27b(+)中,构建出pET27-M重组质粒。生物信息学分析结果显示,M蛋白基因的序列长度为588 bp,编码195个氨基酸残基,推导分子量约为21.88 ku,等电点为9.35;氨基酸序列分析表明,M蛋白富含丝氨酸、苏氨酸以及碱性氨基酸,存在丰富的α-螺旋、β折叠和无规则卷曲;M蛋白不含有信号肽;疏水性大于亲水性;没有跨膜区存在;抗原表位预测显示抗原性良好;结构预测显示,不存在N-糖基化位点,存在7个潜在的O-糖基化位点和15个潜在的磷酸化位点。系统进化树分析显示,IHNV-Sn株与美国分离株同为一簇。     

传染性皮下及造血组织坏死病毒NJ株ORF3基因的原核表达及生物信息学分析

为探究本实验室分离的传染性皮下及造血组织坏死病毒NJ株(IHHNV-NJ)ORF3基因编码蛋白的结构特征,本实验根据ORF3基因序列设计引物,利用PCR方法克隆ORF3基因序列,并构建至原核表达载体p ET-32a(+)中。对成功构建的p ET32a-ORF3重组表达载体进行原核表达,获得49 ku的融合蛋白,符合预期大小。通过生物信息学软件对ORF3基因编码蛋白序列进行分析,结果显示,ORF3基因序列长度为990 bp,编码329个氨基酸;ORF3基因编码蛋白理论分子质量为37 385.2 u,等电点为7.22,为亲水性蛋白;该编码蛋白序列不存在跨膜区、信号肽切割位点;二级结构含有55.9%的α-螺旋、52.0%的β-折叠以及13.4%的β-转角;抗原表位分布较广泛,抗原性强;该编码蛋白序列不存在潜在的N-糖基化位点,存在13个潜在的O-糖基化位点和17个潜在的磷酸化位点。进化树结果表明,NJ株的ORF3基因编码蛋白序列与6株IHHNV序列同源性均高于96%,与厄瓜多尔株同源性最高,为99.7%。研究表明,ORF3基因编码IHHNV衣壳蛋白;O-糖基化位点可参与衣壳蛋白的组装过程及细胞侵染过程,磷酸化位点可参与病毒在对虾细胞内的增殖过程;ORF3编码蛋白序列保守性强,不影响病毒毒力和个体间感染能力。     

急性病毒性坏死病毒魁蚶株IAP-86基因全长cDNA克隆和生物信息学分析

为深入探讨急性病毒性坏死病毒(Acute Viral Necrosis Virus, AVNV)魁蚶株的致病机理以及IAP-86蛋白的功能,从感染 AVNV 的濒死魁蚶外套膜中提取总 RNA,反转录获得 cDNA。根据 NCBI公布的 AVNV 全基因组序列中 ORF86序列设计两对反向套式引物,通过 cDNA 末端快速扩增(RACE)技术获得 ORF865￠端和3￠端的非编码区,拼接获得全长 cDNA 序列。Blast 序列比对显示,该基因与牡蛎疱疹病毒的同源性为100%,与栉孔扇贝 AVNV 的同源性为99%,并存在重叠基因。生物信息学分析预测,该蛋白不含信号肽,不存在跨膜区,最大疏水指数为1.800,最小疏水指数为–3.456。该蛋白存在8个潜在的磷酸化位点(包括5个丝氨酸、1个苏氨酸和2个酪氨酸),存在 1个潜在的 O-糖基化位点,不存在潜在的 N-糖基化位点;其抗原表位主要集中在8?11、14?16、28?39、75?76、88?95、97?100和147?158位氨基酸。推测该株病毒可能为牡蛎疱疹病毒的变异株,并且基因重叠在该类病毒进化过程中可能扮演重要角色。     

大菱鲆促黄体激素受体基因的克隆及其生物信息学分析

通过兼并引物扩增及RACE技术,克隆大菱鲆促黄体激素受体(luteinizing hormone receptor,LHR)基因,分析其相应生物信息学特征和组织表达。结果显示:LHR基因序列全长3 184 bp,编码685个氨基酸,同大西洋庸鲽同源性最高。氨基酸序列分析发现,该基因编码蛋白为疏水性蛋白,相对分子量76.54 ku,理论等电点7.22,存在信号肽序列和跨膜区;亚细胞定位主要在内质网和细胞膜;预测该蛋白含有33个磷酸化位点、5个糖基化位点、6个富含亮氨酸重复序列、7个跨膜保守结构域,二级结构以无规则卷曲为主,三级结构其结构域呈α螺旋状构象;蛋白功能预测表明,LHR主要在辅助、运载、翻译和代谢调节过程中发挥关键作用。组织表达分析表明,LHR基因除在卵巢大量表达外,在其他非性腺组织也有表达,尤其是在肝脏表达较高。上述结果为深入探讨LHR基因在大菱鲆性腺发育过程中的生物学功能奠定了遗传信息基础,也为研究其他海水养殖鱼类生殖调控提供重要参考。     

红笛鲷MAPKK5基因的克隆及生物信息学分析

利用本实验室构建红笛鲷头肾cDNA文库中的MAPKK5基因EST序列,通过RACE PCR技术克隆红笛鲷MAPKK5基因cDNA全长(Ls-MAPKK5,登录号为KM657202)。序列分析结果显示,Ls-MAPKK5cDNA全长2632bp,其中开放阅读框1317bp,可编码438个氨基酸,预测其编码蛋白的分子量为49.29ku,理论等电点为6.12。SignalP 4.0、TMHMM Server 2.0和SoftBerry-Psite预测结果显示,Ls-MAPKK5没有信号肽和跨膜结构,含有22个磷酸化位点。氨基酸序列分析显示LsMAPKK5具有保守的PB1与S_TKc结构域。利用MEGA5.0软件,根据邻位相连法构建MAPKK5系统进化树,结果显示Ls-MAPKK5与雀鲷、吉富罗非鱼该蛋白有较近的亲缘关系。     

拟态弧菌安徽分离株外膜蛋白基因OmpU的克隆测序与生物信息学分析

测序结果显示4株拟态弧菌OmpU基因片段长为849~876bp,编码283~292个氨基酸;拟态弧菌HX4分离株OmpU基因ORF长1 038 bp,编码346个氨基酸。生物信息学分析结果显示,OmpU基因在拟态弧菌安徽分离株之间及其与霍乱弧菌参考株间均具有较高保守性,彼此间核苷酸同源性和氨基酸同源性分别介于82.8%~99.6%和83.3%~100%;拟态弧菌OmpU蛋白N端前22个氨基酸组成信号肽,N末端有1个明显疏水区,并含有多个蛋白激酶磷酸化位点和糖基化位点;OmpU蛋白含有2个明显跨膜域,二级结构中富含β-折叠结构,在蛋白的53~66 aa、185~192 aa、215~219 aa和275~281 aa之间可能存在抗原表位。     

锦鲤疱疹病毒囊膜蛋白ORF59的克隆、分析及其主要B细胞表位区的原核表达

从感染锦鲤疱疹病毒（Koi herpesvirus,KHV）的锦鲤（Cyprinus carpiokoi）肾脏组织中提取DNA,通过PCR扩增了KHVORF59基因。该基因全长411bp,所编码的蛋白包含136个氨基酸,分子量14.3kDa,等电点（PI）6.91,有12个潜在的O糖基化位点。此研究克隆的KHVORF59基因第130位碱基由G突变为A,使其编码的第44位氨基酸由Ala突变为Thr。采用DNAStar程序,在综合分析二级结构柔性区、蛋白的亲水性、表面可能性和抗原性指数的基础上,预测了KHVORF59蛋白主要B细胞表位,并将其区段的编码序列与KHVORF59完整编码序列分别克隆入原核表达载体pET-32a（＋）,构建重组质粒pET32a-ORF59S和pET32a-ORF59C,转入大肠杆菌Rosetta菌株,IPTG诱导表达。SDS-PAGE及WesternBlot分析显示,pET32a-ORF59S可以高效表达,表达的截短KHVORF59蛋白主要以可溶性形式存在,采用HisBindResin填料,层析纯化了该截短蛋白。     

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.