哲罗鲑胰岛素样生长因子-Ⅰ(IGF-Ⅰ)cDNA分子克隆、序列分析及组织表达

采用逆转录聚合酶链式反应(RT-PCR)方法,从哲罗鲑(Hucho taimen)肝脏的总RNA中扩增出胰岛素样生长因子-Ⅰ(IGF-Ⅰ)的c DNA开放阅读框(Open reading frame,ORF)序列,运用软件对其进行生物信息学分析,并利用荧光实时定量PCR技术检测了哲罗鲑成鱼不同组织中IGF-Ⅰ mRNA的表达情况。结果显示,IGF-Ⅰ基因的c DNA开放阅读框为573 bp,编码190个氨基酸,蛋白质等电点为9.21,氨基酸结构由信号肽、B、C、A、D结构域及E肽组成;氨基酸序列与其他鲑科鱼类具有较高的同源性,其中与北极红点鲑的IGF-Ⅰ同源性最高(99.2%);组织表达分析显示,哲罗鲑IGF-Ⅰ mRNA在肝脏中表达量最高,在鳃、前肠中次之,在脑、头肾、脾、心、胃和肌肉等组织中的表达量较低。     

虹鳟pmela基因的克隆及其在不同发育阶段和组织中的表达分析

前黑素小体蛋白a (premelanosome protein, pmela)基因是黑色素合成通路中的关键基因之一，对动物的体色有着重要影响。为探讨pmela基因在虹鳟(Oncorhynchus mykiss)体色变异中的作用，本研究利用cDNA末端快速扩增(rapid amplification of cDNA ends, RACE)技术对pmela基因cDNA全长进行克隆并运用生物信息学方法分析该基因的序列特征，同时采用qRT-PCR比较pmela在野生型虹鳟(虹鳟)、黄色突变型虹鳟(金鳟)和杂交F1代受精期至孵化后3个月不同发育时期及成鱼不同组织中的相对表达量。研究获得pmela基因cDNA全长序列3 476 bp，包含2 532 bp开放阅读框，编码843个氨基酸。序列分析发现，Pmela为疏水性蛋白且存在PKD功能结构域。同源性比对发现，虹鳟Pemla氨基酸序列与红鲑(Oncorhynchus nerka)的同源性高达97.75%；进化分析结果显示，虹鳟与红鲑的亲缘关系最近，与人类(Homo sapiens)和小鼠(Mus musculus)的亲缘关系最远。qRT-PCR结果显示，pmela基因在虹鳟与金鳟胚胎及出膜后各时期均有表达，在受精期至16细胞期中的表达为虹鳟高于金鳟，出膜后该基因在金鳟背部皮肤中的表达均高于虹鳟，且在4细胞期、16细胞期、原肠期、神经期、体节期、心跳期、1 day post-hatching (1 dph)、3 dph、5 dph、7 dph、10 dph、1 month post-hatching (1 M)和2 M时期中，虹鳟与金鳟的表达存在显著差异。在各组织中，pmela基因在虹鳟与金鳟背部皮肤和眼睛中的表达显著高于其他组织。在杂交F1代中，pmela基因在不同发育时期和组织中的表达模式与双亲类似，且在大部分相同时期和组织中的表达与双亲存在显著差异。上述结果表明，pmela基因的表达量高低与虹鳟体色具有一定的相关性，且可能参与其体色的形成过程。本研究结果为进一步研究pmela基因在虹鳟体色变异中的作用提供基础资料。     

魁蚶C型凝集素基因cDNA的克隆及表达分析

通过cDNA末端快速扩增(Rapid amplification of c DNA ends,RACE)技术克隆得到魁蚶(Scapharca broughtonii)C型凝集素(C-type lectin,Sb-Lec1)基因,该基因全长为700 bp,其中,5′-UTR为29 bp,3′-UTR为167 bp,开放阅读框长度为504 bp,编码167个氨基酸,包括长度为23个氨基酸的信号肽序列、129个氨基酸的糖识别结构域(CRD)以及参与二硫键形成的6个半胱氨酸。预测蛋白分子量为19.11 k Da,理论等电点为4.74。多序列比对结果显示,Sb-Lec1基因CRD编码的氨基酸序列与长牡蛎(Crassostrea gigas)、紫贻贝(Mytilus galloprovincialis)和海湾扇贝(Argopecten irradians)C型凝集素的同源性分别为38%~40%、34%~35%和38%~39%,Sb-Lec1基因编码的氨基酸序列与其他物种的凝集素基因具有相似的结构,均含有形成二硫键的4个保守半胱氨酸。系统进化分析结果显示,魁蚶先与贝类聚为一支,再与脊椎动物聚在一起,表明魁蚶Sb-Lec1基因在进化树上的位置与其传统分类所处位置一致。采用荧光定量PCR技术,检测了Sb-Lec1基因在组织中的表达情况,发现其在肝胰腺、血淋巴、鳃、外套膜、闭壳肌、斧足中均有表达,其中,肝胰腺表达量最高。同时,分析了Sb-Lec1基因在鳗弧菌(Vibrio anguillarum)刺激下的mRNA表达量变化情况。结果显示,与对照组相比,菌刺激组Sb-Lec1基因mRNA在各检测组织中的表达量均显著上调(P0.05),随着刺激时间的延长,表达量呈先升高后降低的趋势。本研究表明,魁蚶Sb-Lec1基因在机体免疫防御方面发挥重要功能。     

刀鲚PPARγ基因的cDNA克隆及其应激应答

旨在研究刀鲚(Coilia nasus)过氧化物酶体增殖物激活受体γ(peroxisome proliferator activated receptor, PPARγ)基因的应激调控表达,克隆并获得了刀鲚PPARγ基因的cDNA全长。刀鲚PPARγ基因的cDNA全长1951 bp,开放阅读框1470 bp,预测编码489个氨基酸。刀鲚PPARγ包括4个功能结构域,即A/B区, DNA结合区(DNA binding domain, DBD区),铰链区,配体结合区(ligand binding domain, LBD区)。运用荧光定量PCR (real time quantitative PCR, RT-qPCR)检测刀鲚PPARγ基因在不同组织、运输胁迫和胚胎发育时期的表达。结果显示,刀鲚PPARγ在各组织中均有表达,其中在肝中表达量最高,在脑、肠、心脏、肾、头肾、肌肉中相对高表达,在鳃和脾中微量表达。运输胁迫过程中, PPARγ基因表达显著上调(P0.05),在4 h达到峰值,随后显著降低,但仍高于对照组。PPARγ在胚胎发育时期各时期均表达,其中在受精卵时期高表达,随后表达量急剧降低,并在此后的时期一直处于较低的表达水平。PPARγ基因在应激过程中发挥重要作用,也是胚胎发育过程中重要的基因。本研究为刀鲚的人工繁育和应激调控提供了理论基础。     

镜鲤△6脂肪酸脱氢酶CDNA的克隆与表达

利用RT-PCR和RACE方法克隆得到镜鲤Cyprinus carpio肝脏中高不饱和脂肪酸(HUFA)合成代谢的脂肪酸去饱和酶(△6FAD)基因c DNA全序列。结果表明:镜鲤△6FAD基因c DNA全长为1 446bp,开放阅读框(ORF)为1 332bp,编码444个氨基酸。编码的蛋白序列包含FAD全部特征结构区,包括1个细胞色素b5结构域、2个跨膜区和3个组氨酸簇,与其他鱼类的Δ6FAD氨基酸序列具有69.0%~92.0%的同源性。系统树分析显示:与斑马鱼Danio rerio的亲缘关系最近。实时荧光定量PCR(RT-q PCR)检测发现:脂肪酸延长酶基因在镜鲤肝脏中表达量最高,背部肌次之,在血液中表达最低。本研究结果为进一步研究镜鲤HUFA的合成途径及调控机理提供了基础资料。     

鲢鱼恒定链cDNA的克隆及其分子结构分析

为研究鱼类恒定链(Invariant chain,Ii)的结构与功能,应用RT-PCR和RACE(rapid amplification of cDNAends)法,从脾脏细胞中克隆了鲢(Aristichthys molitrix)Ii基因cDNA,并进行了测序。该基因cDNA全长为1 136bp,其中开放阅读框为699 bp,编码232个氨基酸。比对氨基酸序列发现,鲢与其他物种的Ii链有相同的结构域。将Ii基因cDNA片段和表达不同结构域的DNA片段分别插入真核表达质粒pEGFP-C1,转染真核细胞系COS7,发现鲢Ii跨膜区和胞浆区在细胞内具有定位作用。根据推测的氨基酸序列,分析不同种间Ii的同源性,表明鲢与斑马鱼(Brachydanio rerio)的同源性最高(78%),与人等哺乳动物Ii的同源性较低(30%～40%)。进一步模拟构建和比较不同物种Ii链的3D结构,显示链鱼和小鼠、人和鸡的Ii链结构非常相似,甚至在一些具有重要作用的氨基酸是相同的。上述结果表明,作为重要的免疫分子,不同脊椎动物的Ii链不仅在遗传上具有高度同源性,而且在结构方面仍保持着高度的相似性。     

中华鳖MHCⅡα基因cDNA的克隆及组织表达分析

为了探究中华鳖(Pelodiscus sinensis)的主要组织相容性复合体(major histocompatibility complex, MHC)的结构和功能，本研究利用RACE技术成功克隆获得中华鳖MHCⅡα基因的cDNA全长序列，其长度为1296 bp，开放阅读框(ORF)为807 bp，共编码268个氨基酸，分为信号肽序列、Ⅱ类组织相容性抗原α结构域、IGc1结构域及跨膜结构域4个功能结构域。通过NJ法构建的系统进化树分析显示，中华鳖与西部锦龟(Chrysemys picta bellii)亲缘较近，而与哺乳类、鸟类及鱼类亲缘关系较远。荧光定量PCR结果显示，MHCⅡα mRNA在中华鳖8个组织中均有表达，在脾、心、肝及肠组织中表达水平较高，在肌肉组织中表达量最低。感染嗜水气单胞菌(Aeromonas hydrophila) 12 h后，中华鳖MHCⅡα mRNA在肝和肠组织中的表达显著上调；感染1 d时，在脾组织中的表达显著上调；感染1 d及5 d时，在肾组织中的表达显著上调。研究表明，中华鳖MHCⅡα基因参与了中华鳖免疫反应。     

草鱼褪黑激素受体Mtnr1基因克隆及组织表达分析

为了探究褪黑激素受体(melatonin receptor,Mtnr1)基因在草鱼(Ctenopharyngodon idella)中的表达模式和功能,采用RACE技术从草鱼组织中克隆获得Mtnr1基因的6个亚型(Mtnr1Aa、Mtnr1Ab、Mtnr1Alike、Mtnr1Ba、Mtnr1Bb和Mtnr1C)的cDNA序列,采用ProtParam、TMpred、NetPhos、NetNGlyc、Singal4.1及Phyre2等在线软件对Mtnr1基因的6种亚型进行生物信息学分析。同时,通过实时荧光定量PCR检测6种亚型在不同组织中的表达。结果显示,Mtnr1基因6个亚型cDNA全长分别为2045、2036、2031、2799、2535和2477 bp,分别编码350、351、344、356、347和361个氨基酸。6种亚型均由7个跨膜结构域、NRY结构域、CYICHS结构域和NAXXY结构域及G蛋白偶联受体结合位点组成,同时含有多个磷酸化位点和糖基化位点。Mtnr1基因6个亚型蛋白均由20种氨基酸组成,均属于稳定的亲水性蛋白。氨基酸序列比较分析发现,6种亚型与其他鱼对应的亚型同源性很高,分别为93.1%~99.4%、84.8%~95.2%、82.8%~96.8%、90.1%~97.5%、79.7%~98.3%和90.6%~95.6%。邻接法构建系统进化树显示,6种亚型均与鲤科(Cyprinidae)鱼类聚为一支,与鲤鱼(Cyprinus carpio)、鲫鱼(Carassius auratus)和斑马鱼(Danio rerio)亲缘关系最近。此外,6种亚型mRNA在草鱼肝脏、心脏、鳃、脑、肌肉、前肠、中肠、后肠和肾脏9个组织中均有表达,其中,Mtnr1Aa和Mtnr1Ba基因分别在脑和肾脏中表达量最高,表明Mtnr1Aa和Mtnr1Ba可能在草鱼的神经调节和免疫反应中发挥重要作用。     

草鱼褪黑激素受体Mtnr1基因克隆及组织表达分析

为了探究褪黑激素受体(melatonin receptor, Mtnr1)基因在草鱼(Ctenopharyngodon idella)中的表达模式和功能,采用RACE技术从草鱼组织中克隆获得Mtnr1基因的6个亚型(Mtnr1Aa、Mtnr1Ab、Mtnr1Alike、Mtnr1Ba、Mtnr1Bb和Mtnr1C)的cDNA序列,采用ProtParam、TMpred、NetPhos、NetNGlyc、Singal4.1及Phyre2等在线软件对Mtnr1基因的6种亚型进行生物信息学分析。同时,通过实时荧光定量PCR检测6种亚型在不同组织中的表达。结果显示,Mtnr1基因6个亚型cDNA全长分别为2045、2036、2031、2799、2535和2477 bp,分别编码350、351、344、356、347和361个氨基酸。6种亚型均由7个跨膜结构域、NRY结构域、CYICHS结构域和NAXXY结构域及G蛋白偶联受体结合位点组成,同时含有多个磷酸化位点和糖基化位点。Mtnr1基因6个亚型蛋白均由20种氨基酸组成,均属于稳定的亲水性蛋白。氨基酸序列比较分析发现,6种亚型与其他鱼对应的亚型同源性很高,分别为93.1%~99.4%、84.8%~95.2%、82.8%~96.8%、90.1%~97.5%、79.7%~98.3%和90.6%~95.6%。邻接法构建系统进化树显示,6种亚型均与鲤科(Cyprinidae)鱼类聚为一支,与鲤鱼(Cyprinus carpio)、鲫鱼(Carassius auratus)和斑马鱼(Danio rerio)亲缘关系最近。此外,6种亚型mRNA在草鱼肝脏、心脏、鳃、脑、肌肉、前肠、中肠、后肠和肾脏9个组织中均有表达,其中,Mtnr1Aa和Mtnr1Ba基因分别在脑和肾脏中表达量最高,表明Mtnr1Aa和Mtnr1Ba可能在草鱼的神经调节和免疫反应中发挥重要作用。     

团头鲂黑素皮质素3型受体基因克隆及表达分析

黑素皮质素3型受体(MC3R)系统与动物摄食行为以及能量调控密切相关。为丰富鱼类MC3R相关基础研究,探索鱼类摄食行为与能量调控机制,本研究克隆了团头鲂(Megalobrama amblycephala)mc3r基因,采用分子生物信息学和相对荧光定量PCR方法分别对氨基酸序列保守结构域、组织表达分布和禁食条件下的表达变化等开展分析研究。结果显示,团头鲂mc3r基因编码区全长984 bp,编码327个氨基酸,与现有报道的MC3R氨基酸序列高度相似,具有典型的7次跨膜结构域。组织表达图谱分析表明,mc3rmRNA在下丘脑、垂体、肝脏和卵巢有相对较高的表达。禁食试验结果显示,下丘脑和垂体中的转录变化与周期性摄食信号相关,其中下丘脑mc3r mRNA在禁食72 h和14 d的表达显著上调(P0.05),垂体mc3r mRNA在禁食72 h和禁食72 h后恢复投喂6 h表达量显著升高(P0.05),而肝脏中mc3r mRNA在禁食48 h和14 d的表达量显著上调(P0.05)。此外,对血液皮质醇和血糖的监测结果显示,两者均在禁食14 d有显著变化,其中皮质醇水平显著高于对照组(P0.05),而血糖水平显著低于对照组(P0.05)。综合本研究结果表明,MC3R在鲤科鱼类中具有高度相似的氨基酸序列和结构域;在组织中的转录表达变化与食物摄取有明显相关性,对调控鱼体摄食行为和能量代谢具有重要作用。     

大西洋鲑、三倍体虹鳟和金鳟的肌肉营养成分与品质特性

为了解大西洋鲑(Salmo salar)、三倍体虹鳟(Oncorhynchus mykiss)、金鳟(Oncorhynchus mykiss)3种鱼肌肉营养成分和品质特性,利用生化分析、物性分析方法分析3种鱼肌肉的营养成分、氨基酸和脂肪酸组成、肉色、系水力和质构特性。结果表明,大西洋鲑、三倍体虹鳟、金鳟肌肉的水分质量分数分别为62.91%、67.15%、73.02%,粗蛋白质量分数分别为22.39%、21.03%、22.11%,粗脂肪质量分数分别为14.64%、17.16%、5.11%。3种鱼肌肉的滴水损失、黄色值(b~*)、羟脯氨酸含量、内聚性均显著不差异(P0.05)。3种鱼肌肉的硬度和咀嚼性由低到高依次为大西洋鲑、三倍体虹鳟、金鳟,而pH值的结果则与之相反(P0.05)。大西洋鲑和三倍体虹鳟肌肉的灰分、解冻损失、蒸煮损失、回复性、弹性和红色值(a~*)差异不显著(P0.05),但灰分、蒸煮损失、回复性均小于金鳟肌肉的对应指标(P0.05),弹性和红色值(a~*)则均大于金鳟肌肉的对应指标(P0.05)。大西洋鲑、三倍体虹鳟、金鳟肌肉中必需氨基酸含量占氨基酸总量分别为42.28%、41.84%、41.63%(质量分数),必需氨基酸/非必需氨基酸(EAA/NEAA)比值分别为73.25%、71.94%、71.32%,均符合联合国粮农组织/世界卫生组织(FAO/WHO)对优质蛋白质的评价标准;3种鱼肌肉中均检测到22种脂肪酸,组成丰富,其中不饱和脂肪酸含量较高。综上所述,3种鱼的肌肉都是符合人体营养需求的优质水产品,其中大西洋鲑和三倍体虹鳟肉质接近,且都优于金鳟的肉质。     

IGF-I of Orange Spotted Grouper Epinephelus Coioides: cDNA Cloning, Sequencing and Expression in Escherichia Coli

Insulin-like growth factors I and II (IGF-I and IGF-II) are two highly homologous mitogenic peptides that are expressed ubiquitously and show diverse effects on development, growth, and metabolism. The cDNA encoding IGF-I of a teleost, the orange spotted grouper (Epinephelus coioides) was produced from liver by RT-PCR, and rapid amplification of cDNA ends, RACE. Typically, the deduced 186 amino acid protein contains a signal peptide, B, C, A, D and E domains. On the amino acid level, grouper IGF-I shares 97.3% similarity with black seabream (Sparus macrocephalus) with the differences focusing on the B and C domains. The analysis of the E domain showed that grouper IGF-I belonged to Ea-4 type. When mature amino acid sequence was compared with other vertebrates, it revealed higher similarity with black seabream and halibut, while lower similarity with human and mouse. The expression of IGF-I mRNA in adult tissues was studied using RT-PCR. IGF-I mRNA expression level in the liver was significantly higher than those in the brain and muscles. In other tissues, low amount of IGF-I mRNA expression was also detected. The coding region of IGF-I cDNA for mature IGF-I protein was subcloned into an expression plasmid pTRX and fused with E. coli thioredoxin (Trx). Moreover, we have successfully developed an expression system in E. coli to overproduce recombinant grouper IGF-I. Using western blotting, we found that the fusion protein could blot with antiserum to barramundi IGF-I further confirming the immunoactivity of the recombinant IGF-I.     

Appearance of Infectious Hematopoietic Necrosis Virus in Rainbow Trout,Oncorhynchus mykiss,During Seawater Adaptation

About 7% mortality occurred in rainbow trout, Oncorhynchus mykiss, during seawater adaptation at a marine farm in the South Sea of Korea during the winter of 2014. Most diseased fish showed petechial hemorrhaging of gills and internal fat with enlarged spleen. Although no parasites or bacteria were isolated from the diseased fish, all tissue filtrates produced cytopathic effects (CPEs) in fathead minnow and Chinook salmon embryo‐214 cells. The cell culture supernatant showing CPE contained specific 1527‐bp fragment for the infectious hematopoietic necrosis virus (IHNV) glycoprotein gene by polymerase chain reaction. Their nucleotide sequences shared 98.1–98.2% identities with IHNV RtUi02 isolated from rainbow trout in Korea. This isolate (RtGoH14) was closely related to Korean IHNV isolates of genogroup JRt rather than to those of North American and European genogroups. These results suggest that this IHNV isolate might have been introduced to rainbow trout farm (land‐based culture system) in Korea. This is the first report of IHNV infection in rainbow trout during seawater adaptation in Korea.     

The optimum dietary essential amino acid pattern for rainbow trout (Oncorhynchus mykiss), to maximize nitrogen retention and minimize nitrogen excretion

The optimum dietary essential amino acid (EAA) pattern for a given animal species is considered to be that EAA pattern which results in maximum nitrogen (N) retention for protein growth and minimum N excretion. In a series of two 8-week experiments, we investigated the optimum dietary EAA pattern for rainbow trout (Oncorhynchus mykiss). Experimental diets were fed to quadruplicate tanks of fish, using the equalized satiation feeding method. In the first experiment, we used the amino acid deletion method to arrive at an estimate of optimum dietary EAA pattern for rainbow trout. There were 11 dietary treatments: 1 diet with a control EAA pattern, and 10 other diets with 40% deletions of a single EAA from the control pattern. Based on N utilization data, an estimate of optimum dietary EAA pattern for rainbow trout was made. In the second experiment, we compared this dietary EAA pattern with three other estimates of optimum dietary EAA pattern for rainbow trout, based on 1) amino acid composition of rainbow trout whole-body protein, 2) EAA requirements for rainbow trout published by the National Research Council, and 3) EAA requirements for rainbow trout based on nonlinear regression analysis. Response variables included growth rate, feed efficiency ratio and N retention and excretion. The EAA pattern associated with EAA requirements as published by the National Research Council was found to result in the highest mean N retention and lowest mean N excretion, and so was considered the best estimate of optimum EAA pattern of those compared.     

Cloning,sequence analysis,and expression of tyrp1a and tyrp2 genes related to body colour in different developmental stages and tissues of rainbow trout Oncorhynchus mykiss

Tyrosinase-related protein 1 (tyrp1) and tyrosinase-related protein 2 (tyrp2) genes are important downstream regulatory factors for body colour formation in animals. The present study aimed to explore the relationship between tyrp1a and tyrp2 expression and body colour variation between wild-type (WT) and yellow mutant (YM) rainbow trout. The full-length cDNA sequences of trout tyrp1a and tyrp2 were obtained using rapid amplification of cDNA ends, and the bioinformatic analysis was performed. Quantitative real-time PCR (qRT-PCR) was used to investigate the different expression levels of tyrp1a and tyrp2 in different developmental stages and tissues. The full-length cDNAs of tyrp1a and tyrp2 were 2409 bp and 2219 bp encoding predicted proteins of 522 and 529 amino acid residues, respectively. Both proteins possess six conserved domains, including a signal peptide, an EGF-motif, a Pfam tyrosinase motif, a transmembrane structure, and two copper binding sites (CuA and CuB). Amino acid sequence comparisons showed higher conservation of Tyrp1a and Tyrp2 proteins amongst fishes than amongst other vertebrates, which was further confirmed by phylogenetic analysis. qRT-PCR analysis revealed that tyrp1a and tyrp2 expression levels in WT rainbow trout, and tyrpla in YM rainbow trout, were detected from the fertilised stage to 12 months post hatching (12 M), while tyrp1a did not expressed until the blastula in YM rainbow trout. In addition, the expression levels of both of the genes post hatching were significantly higher than those in the embryonic stages, which showed extremely low expression levels. Additionally, there were extremely significant differences (P < 0.01) in expression at the same stages between WT and YM rainbow trout, e.g., at the blastula, gastrula, 7 days post hatching (7 dph), 10 dph, 2 M, 3 M, 6 M, and 12 M stages for tyrp1a; and at the 4-cell, 16-cell, multicell, blastula, somites, heartbeating, 1 dph, 5 dph, 7 dph, 3 M, 6 M, and 12 M stages for tyrp2. Besides, variable expression levels of tyrp1a and tyrp2 were detected in all tissues; significantly high expression was detected in the dorsal skin and eye compared with that in the other tissues in WT and YM rainbow trout (P < 0.05). These results suggested that the expression level changes of tyrp1a and tyrp2 might be closely related to the variation of rainbow trout body colour, which will enrich our knowledge of the molecular mechanism of skin colour variation in rainbow trout and provide data for research into fish skin colour inheritance and improvement.

     

Occurrence of two distinct molecular species of cathepsin B in carp Cyprinus carpio

ABSTRACT:   We purified cathepsins B₁ and B₂ from the ordinary muscle of carp Cyprinus carpio . The N-terminal amino acid sequences (12 residues) of 29 kDa bands of cathepsins B₁ and B₂ are the same and showed high homology of 75% and 83%, respectively, with the heavy chain of rat and human cathepsins B. Based on conserved sequences of other cathepsins B and the N-terminal amino acid sequences of 29 kDa bands, we cloned carp cathepsin B cDNA. The nucleotide sequence of carp cathepsin B cDNA consists of 1470 bp including a 993 bp open reading frame, encoding a deduced protein of 330 amino acids. The deduced amino acid sequence of carp cathepsin B has similarity of 80% to rainbow trout cathepsin B and of 76–78% to other vertebrate cathepsins B. The sequence of its isoform was also determined during molecular cloning, which has 94.8% similarity with first cloned cathepsin B. They are completely same in N-terminal amino acid sequence of heavy chain, active site and potential N-glycosylation site. This indicates there are at least two kinds of cathepsin B functioning in vivo in carp.     

一株传染性造血器官坏死病病毒的致病性研究

为了对分离于山东某虹鳟养殖场的一株传染性造血器官坏死病毒株(IHNV-Sn1203)进行致病性检测与研究,将该IHNV-Sn1203毒株进行虹鳟鱼苗人工回接感染实验。结果显示,8d内人工感染实验鱼累计死亡率高达100%。收集大批濒死的病鱼样本,制备病理组织切片;利用鲤上皮细胞(EPC)进行细胞感染实验、病毒电镜观察、空斑实验、病毒滴度检测和聚类分析。病理组织切片显示,该病毒可造成虹鳟造血器官广泛性坏死;细胞感染实验结果显示,接种24 h后EPC细胞出现葡萄串状典型细胞病变(cytopathic effect,CPE),72 h后大部分细胞崩解脱落形成网状孔洞;电镜下清晰可见弹状病毒粒子大量存在于细胞质内,其在EPC细胞上的滴度为108.36TCID50/mL,并能形成2~4 mm空斑。对病毒核蛋白氨基酸序列的聚类分析结果显示,该病毒与标准毒株RB-1和WRAC的同源性分别为97%和93%,与国内报道的zyx株具有最高的同源性(99%)。研究表明,IHNV-Sn1203毒株能够在鱼体及敏感细胞中稳定繁殖,产生典型病变,具有较高的病毒滴度,对虹鳟鱼苗有很高的感染性和致死性。