斑点叉尾鮰Cbx基因家族的全基因组鉴定与进化分析

利用Cbx家族基因高度保守的Chromo结构域氨基酸序列作为种子序列,检索斑点叉尾鮰(Ictalurus punetaus)基因组注释的蛋白数据库,共鉴定分离出14个Cbx基因。并在全基因组水平对斑点叉尾鮰Cbx基因家族进行了保守结构域序列、进化、基因结构、染色体定位的系统分析。本研究中鉴定出的斑点叉尾鮰Cbx家族基因蛋白序列与已知的人类、小鼠、鸡、斑马鱼等物种Cbx蛋白序列构建系统进化树。根据系统进化树分析结果,14个斑点叉尾鮰Cbx基因分成两个亚族,5个隶属于Hp1s亚族,9个隶属于PcGs亚族。Cbx基因主要分布于斑点叉尾鮰7条染色体中,且呈不均匀分布。本研究对于后续斑点叉尾鮰Cbx基因家族深入的功能验证及分子作用机制研究具有重要的意义,也为日益丰富的水产基因组资源的挖掘利用提供了参考。     

斑点叉尾鮰套肠症的病原学初步研究

本研究从2例垂死的患败血症并肠套叠的斑点叉尾鮰血液、肝脏及脑组织中分离到2种类型的致病性细菌菌株,经对细菌进行形态观察、生理生化测试和16S rDNA序列分析,分别鉴定为嗜水气单胞菌和荧光假单胞菌;攻毒实验证实它们确为斑点叉尾鮰病鱼的病原。根据药敏实验的结果,及时对病区斑点叉尾鮰进行治疗,连续用药3-5天,发病的叉尾鮰即停止死亡。根据研究结果,文章对斑点叉尾鮰肠套叠发生的原因进行了的分析,提出了不同的观点。     

斑点叉尾鮰源普通变形杆菌的分离、鉴定及药敏特性

【目的】对患病斑点叉尾鮰进行病原菌分离、鉴定及药敏实验,为斑点叉尾鮰肠道坏死病的防控提供参考。【方法】从患病斑点叉尾鮰病灶、肝、脾和肾分离纯化病原菌,经理化特性测定及16S rRNA基因序列分析对其进行鉴定,开展人工感染试验,并利用纸片扩散法进行药敏特性分析。【结果】分离菌株k1为本次引发斑点叉尾鮰病害的致病菌,其对斑点叉尾鮰的LD50为2.82×10~5 CFU/g。菌株k1理化特性与普通变形杆菌Proteus vulgaris基本一致,16S rRNA基因序列与普通变形杆菌相似性最高,综合判定分离菌株为普通变形杆菌。分离菌株k1对环丙沙星、头孢唑林及头孢拉定等12种抗生素高度敏感,对苯唑西林、阿莫西林及痢特灵等7种抗生素耐药。【结论】分离菌株k1是斑点叉尾鮰病原菌,养殖时可选用庆大霉素及氟苯尼考等药物进行防控。     

斑点叉尾鮰鱼油脂成分分析

采用铁锅炼制提取斑点叉尾鮰内脏鱼油,然后加酸使其甲酯化,以气相色谱/质谱法分析鱼油中的脂肪酸.结果斑点叉尾鮰内脏纯油脂中鱼油达到99%.从鱼油中共鉴定出15种成分,有饱和脂肪酸及不饱和脂肪酸,还有少量烷烃类物质.不饱和脂肪酸含量为76.40%,其中多不饱和脂肪酸为18.03%,以C18∶ 2(16.23%)为主,单不饱和脂肪酸为58.37%,以C18∶ 1(54.88%)为主.饱和脂肪酸含量为20.91%,主要有C16∶ 0 (15.84%)和C18∶ 0(4.29%),多是低于C18以上的中长链脂肪酸.因此斑点叉尾鮰油脂可作功能性脂肪酸的重要膳食来源.     

基于微卫星标记的斑点叉尾鮰家系鉴定技术及应用

研究旨在建立准确、高效且经济的斑点叉尾鮰(Ictalures punctatus)家系亲缘鉴定体系, 以期为斑点叉尾鮰的遗传评估及家系育种提供科学依据。选用10对具有较高多态性SSR标记, 建立两个5重PCR反应体系。应用建立的斑点叉尾鮰家系亲缘鉴定体系对来源于13个全同胞家系和8个半同胞家系的333尾个体进行亲权鉴定。结果表明: 10个位点平均等位基因数为9.8、平均观测杂合度0.8591、平均期望杂合度0.8092、平均多态信息含量0.7845; 3种情况下的累积排除概率分别为: 0.99996806、0.99833267和0.99999998; 验证群体的鉴定结果与系谱高度一致, 真实鉴定率达到99.1%, 子代与父母本三者之间配对平均LOD值介于13.30—24.70, 且置信度均达到95%。研究选择的微卫星位点等位基因数目较多, 多态性较高, 可以快速、准确地对斑点叉尾鮰混养群体进行家系鉴定。     

基于重组毕赤酵母的斑点叉尾鮰β-防御素表达

β-防御素是斑点叉尾鮰Ietalurus punetaus抵御病原微生物侵染的首要蛋白质免疫因子,其一级结构包含氨基端24个氨基酸组成的信号肽和羧基端43个氨基酸组成的成熟肽,该成熟肽赋予β-防御素的生物学活性。文中首次构建了产斑点叉尾鮰β-防御素的毕赤酵母Pichiapastoris重组菌株,实现了基于真核表达的斑点叉尾鮰β-防御素的生物合成。首先通过RT-PCR从斑点叉尾鮰皮肤中分离β-防御素成熟肽基因"IPBD",将其与表达载体p PICZαA连接并转入毕赤酵母X-33后,获得重组毕赤酵母菌株;经含1 000μg/m L博来霉素的培养基筛选,获得高拷贝重组菌株。以BMM培养基(无氨基氮源培养基)替代BMMY培养基(含氨基氮源培养基),对重组菌株的发酵培养条件进行优化,确定其产斑点叉尾鮰β-防御素的最适条件为:28℃、250 r/min、1.0%甲醇诱导表达96 h。重组菌株产物经镍离子亲和层析获得分子量为5.98k Da的纯化蛋白,基于MALDI-TOF-TOF的质谱分析证明该纯化蛋白为重组IPBD。抑菌活性测定结果表明重组IPBD对革兰氏阳性的金黄色葡萄球菌Staphylococcus aureus、单增李斯特菌Listeria monocytogenes以及革兰氏阴性的铜绿假单胞菌Pseudomonas aeruginosa的抑菌率分别为69.6%、71.6%和65.8%。本研究为鱼类来源天然小分子抗菌肽的开发提供了可参考的重组DNA技术。     

盐酸氯苯胍在斑点叉尾鮰体内的药代动力学及残留消除规律

在水温(28±1)℃条件下, 以20 mg/kg剂量的盐酸氯苯胍口灌斑点叉尾鮰(Ictalurus punctatus), 采用高效液相色谱-串联质谱法研究盐酸氯苯胍在斑点叉尾鮰体内的药代动力学和残留消除规律。结果显示, 在单次口灌给药后, 盐酸氯苯胍在斑点叉尾鮰血浆中的药时曲线符合二室模型特征, 其药动学方程为C= 7.69e–0.02t+ 0.13e–0.01t–7.82e–0.27t。盐酸氯苯胍在斑点叉尾鮰血浆、肌肉、皮、鳃、肝脏和肾脏中的T_(peak)分别为10.03、15.79、11.10、2.61、12.89、7.87h; C_max分别为5.76 μg/mL、2.91、2.90、3.05、3.04、0.42 mg/kg, 消除半衰期分别为58.63、23.57、35.37、19.74、29.34、43.30h; 药-时曲线下面积AUC分别为326.74 (μg/mL)/h、157.58、183.72、95.09、174.82、29.85 (mg/kg)/h。在连续口灌给药5d后, 停药后盐酸氯苯胍在斑点叉尾鮰肠道中的浓度最高, 肌肉中浓度最低, 盐酸氯苯胍在体内各组织中的消除速度由高到低依次为血浆、鳃、脑、肌肉、皮肤、肝脏、肾脏、肠。若将10 μg/kg作为盐酸氯苯胍的最高残留限量, 在试验条件下, 建议盐酸氯苯胍在斑点叉尾鮰体内的休药期至少应为23d。     

斑点叉尾鮰巨噬细胞移动抑制因子(MIF)基因多态性及表达分析

巨噬细胞移动抑制因子(MIF)是一类主要由T细胞产生、负责机体在环境协迫和炎症反应中进行免疫应答的细胞因子。根据斑点叉尾鮰MIF基因EST序列,应用RACE技术克隆了MIF 3'UTR和5'UTR序列,利用Genome Walking技术克隆了MIF启动子序列,通过特异PCR克隆MIF内含子序列,并对多个体MIF基因组序列进行多态性分析,利用荧光定量PCR研究MIF的组织分布。结果显示,斑点叉尾鮰MIF基因组序列共3 918 bp,其中编码区348 bp,编码115个氨基酸,5'UTR为70 bp,3'UTR为434 bp,启动子为1 382 bp,共有2个内含子,分别为243 bp和1 441 bp。MIF基因组序列中包含4个重复单元序列和60个多态性位点,其中共有6个SNP位于转录因子结合位点上。斑点叉尾鮰MIF mRNA在肠中表达量最高,肌肉中表达量最低。     

氧化油脂对斑点叉尾鮰生长、体色和抗氧化能力的影响

为考察正常及氧化鱼油、豆油、混合油脂(鱼油﹕豆油=1﹕1)对斑点叉尾鮰(Ictalurus punctatus)生长性能、体色和肝脏抗氧化能力的影响, 设计6组等氮等脂饲料, 分别添加6%鱼油、6%豆油、3%鱼油+3%豆油、6%氧化鱼油、6%氧化豆油、3%氧化鱼油+3%氧化豆油, 饲喂6组初始体重(150.5±4.2 g)的斑点叉尾鮰8周, 每组3个重复, 每个重复14尾鱼。结果表明, 摄食6%鱼油、6%豆油、3%鱼油+3%豆油饲料的斑点叉尾鮰在增重率、饲料系数、肌肉组成、体色和肝脏抗氧化指标上均无显著差异(P>0.05); 摄食3组氧化油脂饲料后, 鱼体增重率和肌肉粗脂肪含量降低(P<0.05), 饲料系数和背部、尾部皮肤b*(黄度值)增加(P<0.05), 肝脏丙二醛(MDA)和血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、总胆红素(TBIL)和直接胆红素升高(DBIL)(P<0.05), 肝脏超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、还原型谷胱甘肽降低(GSH)(P<0.05), 而肌肉色度值(L*、a* 、b*)和肌肉水分、粗蛋白质、灰分含量无显著差异(P>0.05)。以上结果表明, 6%氧化油脂(鱼油、豆油或混合油脂)导致斑点叉尾鮰生长性能下降、皮肤黄度增加、肝脏抗氧化能力受损; 豆油可替代斑点叉尾鮰饲料中鱼油的使用, 而不会对生长产生负面影响。     

斑点叉尾鮰LEAP-2和NK-lysin抗菌肽在毕赤酵母中的串联表达

[目的]合成密码子优化的斑点叉尾鮰(Ictalurus punctatus)LEAP-2与NK-lysin成熟肽的串联基因(LN),构建真核重组表达载体p PICZαA-LN,实现LN在毕赤酵母中的重组DNA表达并对其纯化。[方法]参考编码斑点叉尾鮰LEAP-2和NK-lysin成熟肽的c DNA序列,根据毕赤酵母(Pichia pastoris)的密码子偏爱性,优化合成LEAP-2与NK-lysin成熟肽的串联基因LN,两者之间通过α因子信号肽酶切位点对应的核苷酸序列连接;选择表达载体p PICZαA,构建重组表达载体p PICZαA-LN;电转化至毕赤酵母X-33,29℃、p H 6.0条件下,采用1.0%甲醇诱导表达目的蛋白;采用阳离子交换层析对表达产物进行分离纯化。[结果]Tricine-SDS-PAGE分析显示,培养72 h的表达产物经阳离子交换层析,获得了重组体LEAP-2成熟肽、NK-lysin成熟肽和两者的串连表达物;LEAP-2成熟肽的分泌表达效率较NK-lysin成熟肽的高。[结论]实验构建了斑点叉尾鮰LEAP-2与NK-lysin串联基因的真核重组表达载体,并在毕赤酵母X-33中成功表达。     

白斑综合症病毒实时荧光LAMP检测方法的建立及应用

研究利用ESE-Quant tube scanner检测平台, 建立了一套基于环介导等温扩增技术(Loop-Mediated Isothermal Amplification, LAMP)的实时荧光检测方法, 用于白斑综合征病毒(White Spot Syndrome Virus, WSSV)的检测; 并在此基础上, 与巢式PCR、Real-time PCR和其他已发表的4种LAMP方法在检测灵敏度、实际应用方面进行比较. 结果显示, 研究建立的实时荧光LAMP检测方法在63℃恒温反应30min可检测到最低为105倍稀释的基因组DNA模板, 与Real-time PCR检测方法的灵敏度相当, 高于巢式PCR和其他已发表的4种LAMP方法的检测灵敏度; 而且特异性较好, 与传染性皮下及造血组织坏死病毒等5种常见对虾病原DNA均无交叉反应. 通过构建质粒进一步进行灵敏度测试显示, 本研究建立的实时荧光LAMP检测方法最低检测限度为24个拷贝质粒DNA, 检出时间亦为30min. 通过对66份待检样品的检测结果显示, 实时荧光LAMP检测方法的检出阳性率为7.57%, 准确率为100%, 高于其他WSSV的检测方法. 因此, 研究建立的WSSV实时荧光LAMP检测方法, 操作简单, 反应速度快, 特异性好, 灵敏度高, 成本低廉, 可以直观、实时地观察反应的进行情况, 适合对虾养殖现场及诊断实验室的WSSV快速检测.       

Development of reverse transcription loop‐mediated isothermal amplification assay for sensitive and rapid detection of Hosta virus X

The one‐step real‐time turbidity loop‐mediated isothermal amplification assay (RealAmp) was developed to detect Hosta virus X (HVX), the most devastating threat to hosta industry. The reaction was performed in a single tube at 63°C for 15 min, and real‐time turbidimetry was used to monitor the amplification results. Specificity and sensitivity analyses demonstrated that this RealAmp method was sensitive as real‐time TaqMan RT‐PCR and about 100‐fold higher than conventional RT‐PCR with no cross‐reaction with other viral pathogens. Field samples detection showed that HVX could be identified effectively with this method. Overall, this RealAmp assay for HVX detection was simple, specific, sensitive, convenient and time‐saving and could assist in the quarantine measures for prevention and control of the disease caused by HVX.     

Identification of envelope protein orf10 of channel catfish herpesvirus

Channel catfish virus (CCV) is a viral pathogen of fry and fingerling channel catfish and can cause significant commercial loss. Previous studies have shown that the CCV virion contains at least 25 predicted structural proteins, including viral protein 10, which is encoded by the orf10 gene of the CCV. In this paper, the orf10 gene was expressed in Escherichia coli and used to produce a specific antibody. Western blot analysis confirmed that open reading frame 10 is an envelope protein. A viral neutralization assay demonstrated that open reading frame 10 antiserum was able to inhibit CCV infection of channel catfish ovary cells, suggesting that viral protein 10 is likely to play an important role in the CCV infection of channel catfish ovary cells.     

Histophathology and electron microscopy of channel catfish virus in infected channel catfish,Ictalurus punctatus (Rafinesque)*

A histologic and electron microscopic study was made on selected organs from channel catfish Ictalurus punctatus (Rafinesque) fingerlings that were experimentally infected with channel catfish virus (CCV). Histopathology was characterized by necrosis and haemorrhage in kidney and liver, and haemorrhage in the spleen and gastrointestinal tract. Virus replication occurred in nuclei of cells in the kidney, liver and spleen. Intranuclear inclusion bodies consisting of geometric crystalline arrays and lamellar structures were associated with virus replication.     

Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri

Channel catfish Ictalurus punctatus infected with Edwardsiella ictaluri results in 40--50 million dollars annual losses in profits to catfish producers. Early detection of this pathogen is necessary for disease control and reduction of economic loss. In this communication, the loop-mediated isothermal amplification method (LAMP) that amplifies DNA with high specificity and rapidity at an isothermal condition was evaluated for rapid detection of E. ictaluri. A set of four primers, two outer and two inner, was designed specifically to recognize the eip 18 gene of this pathogen. The LAMP reaction mix was optimized. Reaction temperature and time of the LAMP assay for the eip 18 gene were also optimized at 65 degrees C for 60 min, respectively. Our results show that the ladder-like pattern of bands sizes from 234 bp specifically to the E. ictaluri gene was amplified. The detection limit of this LAMP assay was about 20 colony forming units. In addition, this optimized LAMP assay was used to detect the E. ictaluri eip 18 gene in brains of experimentally challenged channel catfish. Thus, we concluded that the LAMP assay can potentially be used for rapid diagnosis in hatcheries and ponds.     

Poly I:C inhibits the expression of channel catfish virus immediate-early gene ORF 1 at early times after infection

Channel catfish virus (CCV) is a herpesvirus that infects channel catfish fry and fingerlings. Previous research has demonstrated that Type I interferons inhibit the expression of immediate-early (IE) genes of some mammalian herpesviruses. However, CCV is distantly related to the mammalian herpesviruses and Type I interferons from higher vertebrates exhibit only 20% similarity to fish interferons. In this work we demonstrate that treatment of channel catfish ovary (CCO) cells, a fibroblast-like cell line, with poly I:C, a known inducer of Type I interferons, results in inhibition of expression of the CCV IE gene ORF 1. Thus, although the genes involved have diverged, the mechanism appears to be conserved. If this paradigm holds true for other CCV IE-Type I interferon interactions, it could have important implications for the impact of CCV on the host immune system.     

Detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Litopenaeus vannamei by ramification amplification assay

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a single-stranded DNA virus that causes developmental and growth abnormalities in Pacific white shrimp Litopenaeus vannamei (also known as Penaeus vannamei). Nucleic acid based methods such as in situ hybridization (ISH) and PCR have been commonly used for IHHNV detection. Ramification amplification (RAM), an isothermal nucleic acid amplification approach, was used in this study to detect IHHNV in L. vannamei. RAM offers many advantages over PCR, including simple procedures and short detection time, and is labor-saving and cost-effective. RAM exponentially amplifies a circular oligonucleotide amplicon (C probe) after a target-specific ligation step through sequential primer extension and strand displacement processes. The conditions of an IHHNV RAM assay were optimized using artificial templates and targets prior to application. Using DNA of IHHNV-infected L. vannamei as targets, results revealed that RAM amplified target DNA with similar sensitivity as PCR. RAM offers competitive levels of speed, simplicity and sensitivity among various pathogen diagnostic methods.     

Cloning and characterisation of a channel catfish (Ictalurus punctatus) Mx gene

A 2.5 kb full-length cDNA clone of a channel catfish (Ictalurus punctatus) Mx gene was obtained using RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) from RNA extracted from the liver of poly I:C stimulated channel catfish. The gene consists of an open reading frame of 1905 nucleotides encoding a 635 amino acid protein. The predicted protein is 72.5 kDa and contains the dynamin family signature, a tripartite GTP binding motif and a leucine zipper, characteristic of all known Mx proteins. The catfish Mx protein exhibited 79% identity with perch Mx and between 71% and 74% identity with the three Atlantic salmon and the three rainbow trout Mx proteins. Mx mRNA was constitutively expressed in channel catfish ovary (CCO) cells, but in higher quantities in response to poly I:C treatment. Mx was induced in channel catfish following injection with channel catfish virus (CCV) and poly I:C.     

Sensitive and rapid detection of Flavobacterium columnare in channel catfish Ictalurus punctatus by a loop-mediated isothermal amplification method

AIMS: To evaluate the loop-mediated isothermal amplification method (LAMP) for rapid detection of Flavobacterium columnare and determine the suitability of LAMP for rapid diagnosis of columnaris infection in channel catfish, Ictalurus punctatus. METHODS AND RESULTS: A set of four primers, two outer and two inner, were designed specifically to recognize 16S ribosomal RNA gene of this pathogen. Bacterial genomic DNA templates were prepared by hot lysis in a lysis buffer. Amplification of the specific gene segments was carried out at 65 degrees C for 1 h. The amplified gene products were analysed by agarose gel electrophoresis and detected by staining gels with ethidium bromide. A PCR assay was also included in this study. Our results demonstrate that the ladder-like pattern of bands from 204 bp specific to the Fl. columnare 16S ribosomal RNA gene was amplified. The detection limit of the LAMP assay was comparable to that of PCR in prepared genomic DNA reactions. In addition, this optimized LAMP assay was able to detect the Fl. columnare 16S ribosomal RNA gene in experimentally infected channel catfish. CONCLUSIONS: The LAMP assay for Fl. columnare detection in channel catfish was established. SIGNIFICANCE AND IMPACT OF THE STUDY: Because LAMP assay is a rapid, sensitive, specific, simple and cost-effective assay for Fl. columnare detection in channel catfish, it is useful for rapid diagnosis of Fl. columnare in fish hatcheries and the field.     

PCR method for detection of largemouth bass virus

A polymerase chain reaction (PCR) method was developed for largemouth bass virus (LMBV). This iridovirus can cause a lethal disease of largemouth bass Micropterus salmoides, but also subclinically infects largemouth bass and other species of fishes. Oligonucleotide primers were designed to specifically amplify the major capsid protein gene of LMBV. The protocol for sample processing and PCR provided a method that was more sensitive than cell culture for detection of LMBV in fish. The specific amplification of LMBV also provided an improved method for confirming the identity of cell-culture isolates presumptively identified as LMBV.