军曹鱼dnd基因cDNA克隆及其在性腺周年发育过程中的表达

本研究通过cDNA末端快速扩增技术(RACE)克隆了军曹鱼(Rachycentron canadum)dnd基因(Rcdnd)的cDNA序列,全长1339bp,其中,5'非编码区59bp,3'非编码区173bp,开放阅读框(ORF)1107 bp,共编码368个氨基酸.Rcdnd氨基酸序列含有1段RNA识别保守基序(R...     

鳜胃蛋白酶原基因cDNA全长的克隆与序列分析

利用RT–PCR和cDNA末端快速扩增法(RACE)克隆鳜(Siniperca chuatsi)胃蛋白酶原基因cDNA全长序列，并对该基因的结构特征和系统进化关系进行了分析。鳜胃蛋白酶原基因cDNA序列全长1367 bp，5′端非翻译区43 bp，3′端非翻译区187 bp，开放阅读框(ORF)1137 bp，共编码378个氨基酸。鳜胃蛋白酶原氨基末端存在信号肽和激活肽序列，序列中含有催化活性必需的2个天冬氨酸残基和构成二硫键的6个半胱氨酸残基。鳜胃蛋白酶原氨基酸序列与其他脊椎动物胃蛋白酶原氨基酸序列的同源性为59.9%～91.2%，表明胃蛋白酶原基因在脊椎动物的长期进化中比较保守。鳜胃蛋白酶原基因的成功克隆不仅为进一步研究该基因的时空表达奠定基础，而且为鱼类胃蛋白酶原的分子特征和进化提供了新的资料。     

圆斑星鲽MHCⅠα基因的克隆与组织表达分析

采用表达序列标签法和cDNA末端快速扩增(RACE)技术,成功获得了圆斑星鲽主要组织相容性复合体MHC Ⅰα的全长cDNA序列.该cDNA全长2171 bp,5'UTR为20 bp,3'UTR为1053bp,开放阅读框(ORF)长度为1098 bp,可编码365个氨基酸,包含信号肽、抗原结合域(α1、α2),IGC类似...     

拟穴青蟹vasa基因cDNA的特征分析与性腺特异表达

vasa蛋白属于DEAD家族蛋白,是一种RNA解旋酶,在生殖细胞的分化中具有重要作用。本研究从拟穴青蟹卵巢中克隆得到vasa基因cDNA全长(将其命名为Sp-vasa),Sp-vasa序列长度为2 944 bp,其中开放阅读框(ORF)长度为1 899 bp,编码632个氨基酸,具有DEAD-box家族蛋白的7个高度保守的结构域。半定量RT-PCR结果显示,Sp-vasa只在卵巢和精巢中特异表达,在其它组织则未检测到表达。由此可见,vasa属于拟穴青蟹性腺特异表达基因。因此,Sp-vasa基因可有望作为有效的分子指标研究拟穴青蟹原始生殖细胞的起源、迁移以及生殖腺的发生。     

刺参vasa-like基因克隆及其在组织中的表达分析

vasa 基因编码DEAD-box 家族成员中一种ATP 依赖的RNA 解旋酶，在生殖系分化过程中发挥着重要的作用。本研究采用同源克隆和cDNA 末端快速扩增技术(RACE)，从刺参(Apostichopus japonicus) 精巢中克隆得到vasa 的全长cDNA 序列。该cDNA 序列全长2 167 bp，开放阅读框1 593 bp，     

真蛸热休克蛋白90基因(HSP90)的克隆及表达

为深入了解真蛸热应激响应机制,实验以真蛸的应激蛋白为研究对象,借助同源扩增和cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术获得真蛸热休克蛋白90(HSP90)的cDNA全序列(2 709 bp),它包含119 bp的5’非编码区(untranslated regions,UTR)、454 bp的3’UTR和2 136 bp的开放阅读框(opening reading frame,ORF),ORF共编码711个氨基酸,推算其分子量为81.5 ku,理论等电点为5.09。同源分析显示,所推导的氨基酸序列高度保守,并且含有HSP90家族特有的5个保守信号序列区域。实时荧光定量PCR分析表明,该基因在所有选取的组织中均有表达,肝组织中表达量最高。     

圆斑星鲽MHC IIB基因结构、多态性及组织表达分析

通过表达序列标签法和cDNA末端快速扩增(RACE)技术,分离和克隆了圆斑星鲽(Verasper variegatus)主要组织相容性复合体(MHC)IIB的全长cDNA序列,该cDNA全长为1144bp,5'UTR(untranslated region)为7bp,3'UTR为450bp,开放阅读框(ORF)长度为687bp,可编码228个氨基酸,包含信号肽、抗原结合域(β1)、IGC区(β2)、跨膜区和胞质区5个结构域。同源分析表明,圆斑星鲽MHCIIB氨基酸序列与其他硬骨鱼具有49%～79%的同源性,与鼠、人、红原鸡和护士鲨的相似性较低,分别为34%、33%、31%和30%。圆斑星鲽MHC ⅡB基因含有5个内含子,与其他硬骨鱼不同,其β2结构域编码区内存在1个109bp的内含子。根据获得的MHC ⅡB基因组序列设计特异性引物,在10尾野生圆斑星鲽中扩增了包括完整内含子1和外显子2的长度约388bp的DNA片段,PCR产物直接测序后发现在270bp的抗原结合域中共有23个位点发生变异,密码子第1位和第2位的变异明显高于第3位。利用荧光定量PCR分析组织表达发现,MHCIIB基因在健康圆斑星鲽9种组织中均有...     

鳜胃泌素与胆囊收缩素基因cDNA全长的克隆与表达特征

为探明鱼类消化液分泌的相关调控因子,采用RACE技术分别克隆了鳜(Siniperca chuatsi)两种肠道激素——胃泌素(gastrin,GAS)与胆囊收缩素(cholecystokinin,CCK)基因的cDNA序列。鳜GAS基因cDNA全长581 bp,5′非翻译区长100 bp,3′非翻译区长145 bp,开放阅读框长336 bp,编码111个氨基酸;鳜CCK具有2种类型:CCK1与CCK2,CCK1 cDNA全长为843 bp,5′非翻译区长60 bp,3′非翻译区长369 bp,开放阅读框414 bp,编码137个氨基酸;CCK2 cDNA全长846 bp,5′非翻译区长112 bp,3′非翻译区长332 bp,开放阅读框403 bp,编码134个氨基酸。鳜GAS与CCK成熟肽C末端具有相似的八肽结构(DYQGWVDF/DYLGWMDF),仅在C末端第3位和第6位氨基酸发生替换。荧光定量PCR分析表明,鳜GAS mRNA主要表达于肠和幽门垂,CCK1与CCK2 mRNA在脑中表达量最高,在肠和幽门垂也有较高表达,表明GAS与CCK同为消化调控因子,而CCK还是神经分泌因子。鳜GAS与CCK mRNA表达贯穿于整个幼体早期发育阶段(孵化后0～22 d),前期表达水平较高,后期表达水平较低,并趋于平稳,GAS与CCK mRNA发育表达水平变化可能与这一时期消化道生长发育旺盛有关。     

鳜脂蛋白脂酶和肝脂酶基因结构与组织表达

为了研究鳜(Sinioerca chuatsi)脂蛋白脂酶(LPL)、肝脂酶(HL)基因结构与功能关系,首先采用RT-PCR及RACE法,克隆得到鳜肝脏LPL与HL基因cDNA全序列.鳜肝脏LPL基因cDNA全长为2089 bp,其中开放阅读框(ORF)为1 548 bp,编码515个氨基酸.鳜HL基因cDNA全长为1 964 bp,其中ORF为1 494 bp,编码497个氨基酸.进化树分析显示,鳜LPL与HL基因与其他脊椎动物的LPL、HL基因各自聚集成簇.对鳜基因组进行PCR获得鳜LPL与HL全基因组DNA序列,与其他脊椎动物基因结构相似,鳜LPL基因由10个外显子和9个内含子组成,全长7 392 bp;鳜HL基因南9个外显子和8个内含子组成,全长8 837 bp.应用Genome Walker方法在鳜克隆得到一段长为1 071 bp的LPL和一段长为2 173 bp的HL基因5'侧翼Ⅸ序列,并利用相关软件预测其中具有多个保守的顺式调控元件.组织表达研究显示与易患动脉粥样硬化的人类LPL不同,鳜LPL基因在所有被检测组织中均有表达:在脂肪组织中大量表达,其次是肝脏、脑、肠道、肌肉,在脾中表达量最低;而鳜HL基因的表达则与人类相似,只在肝脏中表达.本研究将为深入探讨机体脂质代谢调节机制以及LPL、HL在防治动脉粥样硬化中的作用奠定良好基础.     

凡纳滨对虾造血激素基因全长序列分析及其克隆与表达

虾造血激素(astakine,AST)是节肢动物中具有促进造血组织细胞分化和增殖的一种细胞因子。在对白斑综合征病毒(WSSV)感染分子机制的研究中发现,WSSV的一个可能受体蛋白与AST存在特异性结合。为进一步了解WSSV感染与对虾细胞分化之间的关系,本研究应用反转录聚合酶链式反应(RT-PCR)结合cDNA末端快速扩增(RACE)技术,成功克隆了凡纳滨对虾Litopenaeus vannamei造血激素基因(lvast)全长cDNA(GenBank登录号:HM594944),并对该基因进行了生物信息学分析。lvast全长共1846bp,含有1个372bp的开放阅读框,编码124个氨基酸,估算分子量为13.3kD。其编码的氨基酸序列包含一个前动力蛋白(Prokineticin)结构域。基因序列和氨基酸序列同源性比较表明,凡纳滨对虾造血激素(LvAST)与斑节对虾和软尾太平蝲蛄AST同源性较高,与脊椎动物的同源性较低。通过构建包含lvast基因ORF重组表达载体pBAD/gIIIA-lvast,本研究成功表达出与预期相符合的目的蛋白,为进一步研究LvAST的结构与功能提供了理论依据和实验基础。     

剑尾鱼IgZ基因的克隆及疫苗免疫对其在组织中表达的影响

为研究剑尾鱼Ig Z基因序列及其在疫苗免疫后的表达规律,本实验根据已知的EST库设计引物,利用RT-PCR等方法,获得剑尾鱼Ig Z基因c DNA全长序列,并进行生物信息学分析和疫苗免疫后组织表达分析。结果显示,剑尾鱼Ig Z基因全长序列为5 420 bp,包含4个外显子和3个内含子;c DNA序列全长1 527 bp,包含一个1 299 bp的完整ORF框,该基因编码432个氨基酸,预测其编码蛋白的分子量大小为47.48 ku,并具有Ig Z的基本结构,与其他硬骨鱼类Ig Z氨基酸序列一致性为28%~54%。荧光定量PCR分析结果显示,Ig Z基因主要在剑尾鱼的头肾、脾脏和肠中分布,且疫苗免疫后11天内,Ig Z基因在头肾、脾脏和肠组织中均表现为先上升后下降的趋势。头肾中Ig Z基因的表达量在第4天时最高,为对照组的2.12倍;脾脏中Ig Z基因在第4天时呈现最高峰,为对照组的4.65倍;肠组织中Ig Z基因的表达量在12 h时有一个小高峰,第2天时最高,为对照组的11.41倍。本研究获得了剑尾鱼Ig Z基因c DNA全长序列,并对其表达规律进行了初步研究,发现Ig Z在肠道黏膜免疫中具有重要作用,这将为进一步验证其在黏膜免疫中的作用以及剑尾鱼作为疾病研究模式动物和疫苗免疫评价模型奠定基础。     

Vitellogenesis with special emphasis on Indian fishes

Oocyte growth in most oviparous vertebrates including fish is due to the formation of yolk, and eggshell proteins (zona radiata proteins). Zonagenesis leads to the formation of zona radiata proteins in oocytes, which play an important role during oogenesis, whereas vitellogenesis leads to the formation of yolk in oocytes through a series of events during which the yolk precursor protein vitellogenin (Vg) is synthesized and secreted from liver into blood from where it is sequestered into the developing oocytes and thereafter proteolytically cleaved to form yolk proteins (YPs) and finally deposited in the ooplasm. Much research has been done in many fish species with respect to the number and nature of Vg and YPs and their probable functions during fish reproduction. Recent findings of multiplicity of Vg molecules in fishes reject the earlier view of a single-Vg model and have led scientists to explore the functions of individual Vg and their YP derivatives, lipovitellin, phosvitin, and β′-component. Two distinct types of Vg or Vg genes, containing or encoding the three YPs, have been detected in many teleosts. A third unusual, incomplete, phosvitin-poor Vg has been described recently in many fishes. In comparison to much of the information on vitellogenesis in many fishes very little is known for Indian fishes. In India research has been done in a few species such as the catfish, Heteropneustes fossilis and Clarias batrachus, the murrel, Channa punctatus and the Indian major carps, Labeo rohita and Cirrhinus mrigala. Immunological and biochemical analyses suggest the occurrence of multiple forms of Vg and their YP derivatives. The synthesis and incorporation of Vg are regulated by gonadotropin (GTH) and estradiol-17β (E₂). A differential role between estrone (E₁₎ and estriol (E₃) has been demonstrated for Vg synthesis. Enzyme-linked immunosorbent assays (ELISAs) for Vg have been developed to measure plasma Vg. Finally the different roles of Vg1 (HAI) and Vg2 (HAII) on vitellogenesis have been demonstrated. However, more research remains to be carried out in other fish species with respect to the number and nature of Vg and YPs and their genes in order to describe their reproductive functions.     

中华锯齿米虾卵黄蛋白原部分cDNA序列的克隆

卵黄蛋白原(Vitellogenin,Vg)是广泛地存在于卵生脊椎和无脊椎动物体内的雌性特异性蛋白,是卵黄蛋白(vitellin,Vn)的前体,本文通过同源克隆的方法从成熟中华锯齿米虾的卵巢中克隆到了一条长474 bp的Vg cDNA序列。用DNAMAN中的Blast功能将此片段与罗氏沼虾(Macrobrachium rosenbergii)、高背长额虾(Pandalus hypsinotus)的Vg部分序列进行比对,结果发现中华锯齿米虾与罗氏沼虾和高背长额虾的同一性分别为64%与62%。此片段为中华锯齿米虾Vg cDNA全长序列的克隆及其表达奠定了基础。     

Purification and development of ELISAs for two forms of vitellogenin in Indian walking catfish, <Emphasis Type="Italic">Clarias batrachus</Emphasis> (L.)

Two forms of vitellogenin (Vg: Vg1 and Vg2) were purified from the plasma of estradiol-17β (E₂)-treated Indian walking catfish, Clarias batrachus, by gel filtration and adsorption chromatography. Native Vg1 and Vg2 had apparent molecular masses of 375 and 450 kDa, respectively, and both Vgs resolved into two similar major bands (95 and 67 kDa) in SDS-PAGE under reducing condition. Polyclonal antisera raised against each form of Vg were absorbed with a combination of hypophysectomized male catfish serum proteins and alternate Vg to ensure specificity. Immunological analyses verified the presence of Vg1 and Vg2 in the plasma of female catfish. Homologous ELISAs were developed for Vg1 and Vg2 using their respective harvested antisera, which exhibited the detection limit of 100 ng ml^?1 for Vg1 and 40 ng ml^?1 for Vg2, and low level of cross-reactivity (not parallel to the standard) was found with alternate Vg in each assay. Treatment of male catfish with E₂ induced both Vgs showing a proportionate ratio of Vg1 to Vg2 at 5.6:1. Plasma concentrations of both Vgs measured by ELISAs at different reproductive phases of field collected female catfish increased in accordance with the ovarian development, keeping the proportionate ratio of Vg1 to Vg2 at about 2:1 in fish undergoing vitellogenesis during prespawning period and 1:20 during spawning period, suggesting that Vg1 may be the major Vg to contribute in yolk formation, whereas Vg2, besides its role in yolk formation, may facilitate other physiological functions. The present study, thus, demonstrates the occurrence of two unequally synthesized Vgs in the catfish.     

微卫星标记在RR-B系剑尾鱼遗传监测中的应用

采用49对微卫星引物对RR-B系剑尾鱼和红眼红体的非选育系剑尾鱼进行PCR扩增，有46个微卫星座位能获得稳定的扩增产物，7个微卫星座位能区分RR-B系与非选育群体剑尾鱼。7个微卫星座位在选育系剑尾鱼为单态纯合，而在非选育群体具有多态性，座位Msa014鉴定RRB系剑尾鱼排除概率最高，为98.75%，座位Msd003排除概率最低达到了87.50%，其余5个座位排除概率介于两者之间。为方便今后的遗传监测，对RR-B系剑尾鱼样品的7个微卫星座位进行了测序，确定了其大小和具体的微卫星结构。本实验建立了RR-B系剑尾鱼分子检测方法，为实验动物剑尾鱼的遗传监测奠定了基础。图4表3参23     

抗鲤疱疹Ⅱ型病毒卵黄抗体制备及其功能鉴定

为制备抗鲤疱疹Ⅱ型病毒(CyHV-2)的卵黄抗体,探索防治异育银鲫(Carassius auratus gibelio)鳃出血病的新方法和途径,本研究利用原核表达系统产生具免疫原性的重组CyHV-2-ORF72衣壳蛋白,纯化后免疫蛋鸡;二次免疫后采用间接ELISA法抽检免疫蛋的特异性卵黄抗体(IgY)含量,收集抗体效价达到1∶8000的免疫蛋。将免疫蛋分成全蛋组与蛋黄组,采用微包膜和冷冻干燥技术,分别制成全蛋粉或蛋黄粉,利用Western blot和ELISA技术检测全蛋粉和蛋黄粉中的IgY特异性及效价。将蛋黄粉稀释液与CyHV-2混合后孵育鲤鱼上皮瘤细胞,进行抗体中和试验。以0.15%和0.30%的添加量分别将抗CyHV-2-ORF72的免疫全蛋粉拌入商品饲料制成功能性饲料,投喂已发病的两个池塘的异育银鲫进行生产性治疗试验。结果显示,全蛋粉和蛋黄粉中的IgY均能特异性地结合CyHV-2-ORF72衣壳蛋白,效价达到1∶8000~1∶16000;抗CyHV-2-ORF72的IgY与CyHV-2具有较好的亲和力,中和活性高于鼠抗ORF72血清;两个池塘异育银鲫成活率分别达到74.3%和87.6%,远高于对照池的28.3%和31.5%。综上所述,本研究制备的抗CyHV-2-ORF72卵黄抗体可特异性地中和CyHV-2病毒,具较显著的免疫保护作用。     

Deduced primary structures of three types of vitellogenin in mosquitofish (Gambusia affinis), a viviparous fish

Three distinct forms of vitellogenin (Vg), 600 kDa VgA and VgB and 400 kDa Vg, were discovered biochemically in estrogen treated female plasma. By sequencing of the three Vg cDNAs, the VgA and VgB were recognized as complete Vgs having all yolk protein (YP) domains, and the 400 kDa Vg was thought to be phosvitinless (Pvl) Vg lacking phosvitin (Pv) domain.     

剑尾鱼在检测细菌毒力方面的应用

用１８株从鱼类所分离的细菌攻毒剑尾鱼,结果显示细菌对剑尾鱼的毒力与回归感染或攻毒敏感鱼的毒力结果较为一致,另外,用剑尾鱼感染病原菌的适宜途径为背肌注射,与原代比较,近交高代剑尾鱼个体之间对病原菌的反应较为一致,感染后死亡时间相对集中。水生实验动物剑尾鱼在检测鱼类细菌的毒力方面有较好的应用前景。     

Oocyte growth in temperate basses: multiple forms of vitellogenin and their receptor

A full-length cDNA encoding a vitellogenin (Vg) receptor (VgR) was isolated and sequenced from a white perch, Morone americana, ovarian cDNA library. The perch expresses a single VgR that is structurally classified as an LR8 type (without an 0-linked sugar domain) and appears to be the only species observed to possess no splice variant of the receptor. We proposed a hypothetical model for ovarian lipid transfer in Morone species, one in which the `single VgR' transports Vg and other lipoproteins via a selective endocytotic pathway based on ligand affinity, in conjunction with a lipase-mediated pathway of fatty acid transport.