共查询到18条相似文献，搜索用时 281 毫秒
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根据相近物种的同类基因设计引物,从凡纳滨对虾Litopenaeus vannamei肌肉组织中克隆获得原肌球蛋白基因（TPMS）,长度为901bp,其中包含长度为852bp的完整开放阅读框,编码原肌球蛋白分子量为32．8kDa;RT—PCR结果显示,原肌球蛋白在心、肝胰腺、胃、鳃、肠和肌肉组织中均有表达,在肌肉中表达量最高,在鳃中表达量最低;将原肌球蛋白基因构建原核重组表达载体TPMS—pET30a,转化受体菌株BL21（DE3）并利用IPTG诱导后能够大量表达,蛋白分子量为38．2kDa;经优化,IPTG的最适诱导浓度为0．05mmol／L,最适诱导时间为4h;经过亲和纯化后,能够得到纯度90％以上的重组表达蛋白。 相似文献
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中国明对虾酚氧化酶原基因cDNA的克隆与表达特征 总被引：3，自引：0，他引：3
利用3′和5′RACE技术从中国明对虾血细胞中克隆了一个酚氧化酶原基因FCproPO,FCproPO基因cDNA全长为2311bp,其中开放阅读框2061bp,编码687个氨基酸,预测分子量为78.71ku。推测的序列与凡纳滨对虾同源性为84%,与日本囊对虾同源性为71%。RT-PCR实验结果表明,FCproPO在血细胞中的相对表达量最高,在心脏和精巢中几乎不表达。序列分析发现FCproPO含有两个保守的铜离子结合位点;进化分析发现FCproPO与斑节对虾、日本囊对虾、凡纳滨对虾等海水虾的酚氧化酶原亲缘关系最近。该基因在被鳗弧菌和对虾白斑病毒(WSSV)感染后的对虾肝胰腺中的表达量显著增加,并具有不同的时空表达趋势,提示中国明对虾FCproPO基因在免疫反应中具有重要作用。 相似文献
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β-胸腺素(β-thymosin)是一类高度保守的功能多肽,在伤口愈合、血管生成、抗菌和抗病毒免疫等生理活动中起重要的调控作用。本研究从斑节对虾(Penaeus monodon)中成功克隆了一种β-thymosin cDNA序列(β-thymosin 5),其全长为1495 bp,开放阅读框(open reading frame, ORF)为615 bp,编码204个氨基酸。多重序列比对和系统进化树分析结果表明Pmβ5与对虾属的日本囊对虾(Penaeus japonicus)和凡纳滨对虾(Penaeus vannamei)的胸腺素β5聚为一支,其中与日本囊对虾的胸腺素β5同源性最高,相似性高达77%。组织分布研究显示Pmβ5mRNA在肝胰腺和淋巴等免疫相关组织中的表达量明显高于其他组织。在灭活的副溶血弧菌(Vibrio parahemolyticus)刺激下, Pmβ5基因的表达量显著升高(P0.05),这说明Pmβ5参与了对虾的抗菌免疫反应。此外,用原核表达技术获得了Pmβ5的体外重组蛋白,抗菌实验(牛津杯法)检测证明, Pmβ5对哈维氏弧菌、溶藻弧菌、副溶血弧菌和枯草芽孢杆菌均有抗菌活性。 相似文献
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凡纳滨对虾精氨酸激酶的多克隆抗体制备及组织特异性表达分析 总被引：1，自引：0，他引：1
精氨酸激酶（arginine kinase,AK）在调节无脊椎动物磷酸精氨酸与ATP之间能量平衡的过程中具有重要作用。在前期工作基础上,设计特异引物,以凡纳滨对虾肌肉组织cDNA为模版,克隆得到了1 071 bp的凡纳滨对虾精氨酸激酶的完整开放阅读框;将它克隆到原核表达载体pGEX-4T-2上,转化BL21 (DE3) 菌株,经诱导后表达出GST-AK融合蛋白。以纯化的融合蛋白作为抗原免疫小鼠,制备多克隆抗血清,经检测该抗原与抗体识别良好。利用得到的抗体对凡纳滨对虾AK在蛋白质水平上的特异性表达进行分析发现,精氨酸激酶在对虾的肌肉、心脏、神经、血细胞和胃组织中具有高的表达量,而在眼柄、肝胰腺、鳃和皮肤组织中表达量较低。为进一步研究精氨酸激酶在对虾体内的功能奠定了基础。 相似文献
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为研究Akt基因在中华绒螯蟹蜕壳前后肌肉生长过程中的功能,应用RACE技术克隆得到编码中华绒螯蟹Akt(命名为Es Akt)的全长为2 200 bp的c DNA序列,包括159 bp的5′非翻译区(5′-UTR)、496 bp的3′非翻译区(3′-UTR)和长度为1545 bp编码514个氨基酸的编码序列。蛋白质结构域分析显示Es Akt含有丝氨酸/苏氨酸蛋白激酶家族的3个特征性保守结构域。多序列比对和系统发育分析显示,Es Akt与中国明对虾、凡纳滨对虾的Akt序列一致性都为0.889,在系统发育树中节肢动物Akt形成一个分支。应用荧光定量RTPCR技术分析Es Akt在性成熟中华绒螯蟹各组织及幼体不同蜕壳时期不同部位肌肉组织中转录水平上表达量的变化。结果显示,Es Akt在性成熟个体的肝胰腺、眼柄、表皮、卵巢、精巢、心脏、螯足、鳃、三角膜等组织中均有表达,其中卵巢、眼柄和精巢中表达量较高,肝胰腺中表达量最低。在幼体不同蜕壳时期不同部位的肌肉中,Es Akt表达量变化不同,步行足肌肉组织中Es Akt m RNA无显著的统计学差异。腹部肌肉组织中Es Akt m RNA水平在蜕壳前晚期D3~D4期表达量显著高于蜕壳后A~B期和蜕皮间期C期。螯足肌肉在蜕壳前晚期D3~D4期急剧下调,蜕壳后A~B期开始表达量显著升高,直至蜕皮间期C期。研究表明,Es Akt在中华绒螯蟹蜕壳过程中不同部位肌肉组织中的表达量变化与蜕壳周期密切相关,说明Es Akt参与中华绒螯蟹蜕壳诱导的肌肉萎缩、生长及重建过程。 相似文献
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为探究饥饿胁迫对日本囊对虾生长、免疫和能量代谢的影响，在25℃条件下将体质量(0.8±0.06) g的日本囊对虾置于多层多格的格栅式养殖装置中，每格放置1尾个体进行0、3、6、9、12 d的饥饿胁迫，分别检测各时间点胃、肝胰腺和肌肉组织中各种指标的变化情况。试验结果表明：饥饿胁迫下，日本囊对虾生长性能显著降低；各组织中过氧化氢酶活性均呈现先升后降的趋势；酸性磷酸酶、碱性磷酸酶活性在各组织中均随饥饿时间的延长呈显著下降的趋势(P<0.05);超氧化物歧化酶的活性变化呈现出明显的组织特异性，胃组织中的超氧化物歧化酶活性在饥饿胁迫前期呈现上升趋势，并在饥饿3 d达到最高值，随着饥饿时间的延长显著下降，肝胰腺和肌肉组织中的超氧化物歧化酶活性则随着饥饿时间的延长呈显著下降的趋势；饥饿胁迫下日本囊对虾各组织中钠钾ATP酶和钙镁ATP酶活性均呈现先升后降的趋势，且均在胁迫的3 d达到最高值。本试验结果表明，饥饿胁迫不仅会显著降低日本囊对虾的生长性能，在不同胁迫时期对其各组织中免疫相关酶及ATP酶活性也有显著影响。 相似文献
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不同蛋白水平饲料对斑节对虾家系生长及生长相关基因mRNA表达量的影响 总被引：1，自引：0，他引：1
研究了选育出的生长速度不同的9个斑节对虾(Penaeus monodon)家系在蛋白含量为34%、38%和42%饲料的饲喂下,其生长、生长激素基因(GH)、组织蛋白酶-L(Cathepsin-L)、胰蛋白酶(Trypsin)等基因的表达量及生长和基因表达量之间的相互关系。结果表明:1)斑节对虾的生长、3种基因的表达量与饲料中蛋白质含量均呈显著的正相关关系(P0.05);2)随着饲料蛋白质含量的增加,不同斑节对虾家系之间的生长差异和3种基因表达量的差异减小,相同斑节对虾家系在不同蛋白含量饲料的饲喂下,其特定生长率和基因表达量均具有显著性的差异(P0.05);3)选育的9个家系斑节对虾的生长在家系和蛋白质含量这两个因素间没有显著性的交互作用(P0.05),但是肝胰腺和肠道中3种基因的表达量在家系和蛋白质含量这两个因素间具有极显著的交互作用(P0.01),其中,饲料蛋白质含量因素对GH和Trypsin两个基因表达量的贡献率是家系因素的1.31~3.66倍,而饲料蛋白质含量因素对Cathepsin-L基因表达量的贡献率是家系因素的0.06~0.54倍。 相似文献
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为研究酚氧化酶原激活因子(prophenoloxidase-activating factor,PPAF)在凡纳滨对虾感染传染性皮下及造血组织坏死病毒(IHHNV)过程中所起的免疫作用,实验采用逆转录聚合酶链式反应和cDNA末端快速扩增技术克隆了凡纳滨对虾PPAF基因的全长cDNA序列,并通过实时荧光定量PCR技术分析了该基因在正常凡纳滨对虾和感染IHHNV凡纳滨对虾不同组织的表达情况。结果显示,克隆获得了1 986 bp凡纳滨对虾PPAF基因cDNA全长序列(GenBank登录号JQ684529),其中含有一个1 629 bp开放阅读框(ORF),两翼分别存在21 bp(5'端)和336 bp(3'端)的非翻译区。该基因的开放阅读框共编码542个氨基酸,氨基酸序列269~517处存在功能结构域——丝氨酸蛋白酶结构域,氨基酸序列494~502区域存在一个ALPHA-2巨球型免疫蛋白功能位点,316~321区域有一个组氨酸酶活性位点;定量PCR分析发现,该基因无论在正常对虾还是感染IHHNV对虾的心脏、肝胰腺、肠、胃和肌肉中的表达量均较低,在血液和鳃腺中的表达量明显高于其他组织,但感染IHHNV对虾不同组织中PPAF基因的表达量均低于正常对虾相应组织中的表达量。定量检测PPAF基因在对虾鳃腺组织中的表达情况显示,对虾感染IHHNV后,PPAF基因的表达量急剧降低,3 h时至最低,之后表达量逐渐上升,48h时达最高值。研究表明,IHHNV可抑制PPAF基因的表达,因而抑制酚氧化酶原免疫系统的有效激活,或是其感染对虾并在对虾组织内增殖的原因之一。 相似文献
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为探明转化生长因子-β (TGF-β)超蛋白家族的成员之一骨形态发生蛋白7 (BMP-7)基因在中国明对虾肌肉发生和生长过程中的表达模式，通过cDNA末端快速扩增技术得到了中国明对虾BMP-7基因(FcBMP-7基因)2003 bp的全长cDNA,包含466 bp的5′非翻译区和295 bp的3′非翻译区，1242 bp的开放阅读框共编码413个氨基酸。利用实时荧光定量PCR技术分析FcBMP-7基因在中国明对虾幼体原肠期、无节幼体期、溞状幼体期、糠虾幼体期、仔虾期5个不同发育时期肌肉组织中的表达规律，结果显示，该基因在幼体发育的各时期均有表达，原肠期表达量最低，从无节幼体期开始表达量显著增加(P<0.05),糠虾期表达水平最高，表明FcBMP-7基因参与了幼体肌肉的发生过程。此外，进一步分析FcBMP-7基因在中国明对虾2种规格幼虾[(3.95±0.08) g和(1.55±0.12) g]的8种组织中的表达模式，结果显示，FcBMP-7基因具有组织表达广泛性，且两组间的大部分组织的表达量均存在显著差异(P<0.05)。试验结果表明，FcBMP-7基因可能作为调节因子参与了... 相似文献
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中国明对虾C 型凝集素基因(Fclectin)的重组表达及活性分析 总被引：1，自引：1，他引：0
研究拟通过分析对虾C型凝集素的活性特点,探讨其在对虾先天免疫应答过程中的潜在功能以及在养殖生产实践中的应用。实验利用原核表达系统对中国明对虾C-型凝集素基因的两个串联的糖识别结构域(carbohydrate recognition domain,CRD)进行了重组表达,并通过纯化复性获得了重组目的蛋白(rFclectin-CRD1和rFclectin-CRD2)。活性分析结果显示,重组目的蛋白对多种病原菌有凝集和抑制生长的作用,并且具有Ca2+依赖活性;其凝集活性可被半乳糖、肽聚糖、脂多糖等多种病原相关分子模式所抑制,研究结果证实,Fclectin是一种典型的C-型凝集素,它可能作为中国明对虾先天免疫中重要的模式识别受体,在一定程度上参与了机体应答病原微生物的防御过程。 相似文献
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蟹类原肌球蛋白基因的克隆与表达 总被引：4，自引：0，他引：4
原肌球蛋白是甲壳类动物的主要过敏原之一。本文通过分子生物学技术方法,分别克隆得到锯缘青蟹(Scylla serrata)、中华绒鳌蟹(Eriocheir sinensis)和三疣梭子蟹(Portunus trituberculatus)的原肌球蛋白基因序列。测序结果表明,三个基因的序列长度均为855 bp,编码284个氨基酸残基,三者氨基酸序列同源性为99.3%。三种蟹的原肌球蛋白基因序列与GenBank中其他甲壳类动物的原肌球蛋白具有很高的同源性。将锯缘青蟹的原肌球蛋白基因与pGEX-4T-3载体连接后,经IPTG诱导得到分子量约为61 kDa的融合表达蛋白。通过与甲壳类过敏患者血清的免疫印迹反应,证实融合表达的原肌球蛋白具有过敏原性,表明原肌球蛋白是蟹类的主要过敏原之一。该融合蛋白有望用于甲壳类食物过敏诊断试剂的开发与应用。 相似文献
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In vitro assembly of Penaeus monodon densovirus (PmDNV)‐like particles produced in a prokaryote expression system 
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下载免费PDF全文 Rapee Sinnuengnong Pongsopee Attasart Duncan R Smith Sakol Panyim Wanchai Assavalapsakul 《Aquaculture Research》2017,48(9):4975-4981
Viral diseases are a significant problem in the shrimp aquaculture industry as outbreaks can cause significant mortality and economic loss. While it has been shown that triggering the shrimp RNA interference pathway through dsRNA is a potentially viable treatment pathway, this approach is hampered by the lack of a suitable delivery mechanism. Virus‐like particles (VLPs), which are structurally similar to native viruses but lack the genetic material, could possibly be developed as a delivery vehicle. To generate a candidate VLP, the Penaeus monodon densovirus (PmDNV) capsid protein was cloned with an added histidine tag and expressed in an E. coli expression system. While the protein was expressed in inclusion bodies, the recombinant PmDNV capsid protein could be dissolved and subsequently purified by nickel affinity column chromatography. The formation of VLP from this purified rPmDNV capsid protein was investigated by transmission electron microscopy, and PmDNV‐VLPs were observed that looked similar to the native PmDNV virion. Our results suggest that the PmDNV‐like particle could be promisingly applied towards vaccination and that this PmDNV‐like particle can potentially serve as a system for delivery of nucleic acids to trigger innate immunity in shrimp. 相似文献
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Enhancement and confirmation of white spot syndrome virus detection using monoclonal antibody specific to VP26 下载免费PDF全文
下载免费PDF全文 Akapon Vaniksampanna Siwaporn Longyant Pradit Wangman Paisarn Sithigorngul Parin Chaivisuthangkura 《Aquaculture Research》2017,48(4):1699-1710
A portion of the VP26 gene (VP26F109) encoding a structural protein of white spot syndrome virus was expressed, purified by SDS‐PAGE and used for immunization of Swiss mice for monoclonal antibody (MAb) production. Three groups of MAbs specific to different epitopes on VP26 were selected; these MAbs can be used to detect natural WSSV infection in Penaeus vannamei using dot blotting, Western blotting or immunohistochemistry without cross‐reaction with other shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was ranged 7–14 fmole per spot of the rVP26F109 as determined using dot blotting. A combination of three MAbs specific to VP26 with MAbs specific to VP28, VP19 and ICP11 increased the detection sensitivity of WSSV during early infection. Therefore, the MAbs specific to VP26 could be used to confirm and to enhance the detection sensitivity for WSSV infection in shrimp with various types of antibody‐based assays. 相似文献
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The macrophage migration inhibitory factor(MIF)is an important proinflammatory cytokine that mediates both innate and adaptive immune responses.In this study,we identified a homolog of MIF in the Pacific white shrimp Litopenaeus vannamei.The MIF cDNA contained a 363-bp open reading frame encoding a 120-amino acid protein with a calculated molecular mass of 13.442 kDa and a theoretical isoelectric point of 6.57.The L.vannamei MIF shared high amino acid identity with MIFs of other invertebrates.Tissue distribution analysis by quantitative real-time polymerase chain reaction(qRT-PCR)revealed that the L.vannamei MIF was abundantly expressed in the blood,heart,and hepatopancreas,was moderately expressed in the gill,and was weakly expressed in the muscle and intestine.Furthermore,in order to gain a basic understanding of the role of MIF in the shrimp immune response against viral infection,its mRNA expression was determined in the hepatopancreasofL.vannameiatdifferentstagesafter whitespotsyndromevirus(WSSV)challenge usingqRT-PCR.The result indicated that the expression of MIF was significantly upregulated after WSSV injection,suggestingthatMIFmaybeinvolved in theresponsetoviralinfection in shrimp. 相似文献
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Differential expression of proteins in the gills of Litopenaeus vannamei infected with white spot syndrome virus 总被引：1，自引：0，他引：1
Pedro A. Valentim-Neto Ana P. M. Fraga Maria R. F. Marques 《Aquaculture International》2014,22(5):1605-1620
Determination of differentially expressed protein profile is necessary to understand the host response to viral infection. Proteomics can be applied as a tool to examine white shrimp Litopenaeus vannamei molecular responses against white spot syndrome virus (WSSV) infection, thus enabling development of effective strategies to reduce their impact on farms. In the present study, specific pathogen-free shrimp was tested against WSSV infection under several time intervals. Shrimps were submitted to a viral load of with 5.5 × 106 viral copies in 100 μL/shrimp. The monitoring of infection was performed in intervals of 6, 12, 24, 48 and 72 h after infection. The analysis was realized using 2-DE, and differentially expressed proteins were identified by MALDI-TOF mass spectrometry (MS) peptide mass fingerprint (PMF). Between the differentially expressed proteins found in the infected animals, the most important were identified as caspase-2, ubiquitin and F1-ATP synthase. They are interesting candidates for biomarkers because could be related to the beginning of apoptosis process. The differentially expressed protein profile creates a new paradigm in the analysis of L. vannamei shrimp molecular response to WSSV infection and in virus–host relationship. Furthermore, it proposes potential biomarkers that allow strategies both selecting less susceptible individuals and reducing the impact of viruses on farms. 相似文献
18.
Effects of dietary lipid sources on growth and fatty acid composition of juvenile shrimp, Litopenaeus vannamei 总被引：2，自引：0，他引：2
This experiment was conducted to evaluate the effects of dietary lipid sources on the growth performance and fatty acid (FA) composition of juvenile shrimp, Litopenaeus vannamei. Six isoenergetic and isonitrogenous semi‐purified diets containing casein, solvent‐extracted soybean meal and gelatin as protein sources, were supplemented with 60 g kg−1 of lipid sources. The lipid sources included: pollack fish oil (PO), pork lard (PL), soy oil (SO), peanut oil (PN), rapeseed oil (RO) and a mixture of pollack fish oil and soy oil (POSO 1 : 1 w/w). Each diet was fed to juvenile shrimp (0.10 g average weight) four times daily in triplicate tanks to apparent satiation (feeding ratio was about 8%) for 8 weeks. At the end of the experiment, weight gain, specific growth rate and protein efficiency ratio were significantly higher for shrimp fed the diet containing PO and the POSO mixture oil than the other lipid sources. The nutritional values of SO, RO, PN and PL were similar. Shrimp fed on PO, mixture oil of POSO and SO had better survival rates than the other lipid sources, and shrimp fed the PL had the lowest survival rate. There were significant differences in lipid contents of whole body and hepatopancreas amongst the dietary treatments; however, lipid contents of tail muscle were not significantly affected by the dietary lipid sources. Shrimps fed POSO diet had higher protein content in whole body than those fed the other lipid sources, and shrimp fed PO diet had highest crude protein content of the tail muscle. A high correlation was found between dietary FA composition and FA composition of whole shrimp. FA composition of the whole body was generally affected by dietary lipid sources, especially dietary unsaturated FA. 相似文献