共查询到20条相似文献，搜索用时 343 毫秒
1.
Toll受体蛋白(Toll receptors)是一类重要的模式识别受体,在无脊椎动物先天性免疫系统中发挥重要作用。文章对斑节对虾(Penaeus monodon)新型Toll9受体基因(Pm Toll9)进行了研究:以人源胚胎肾细胞(HEK293T)成功构建体外细胞免疫模型,通过免疫印迹方法证实Pm Toll9重组真核蛋白可在HEK293T中成功表达。双荧光素酶报告系统检测发现在200 ng转染浓度处Pm Toll9对NF-κB报告基因的激活效果显著。q RT-PCR数据证明PmToll9成功激活HEK293T细胞Toll-like receptor(TLR)信号通路,促进通路下游髓样分化因子(My D88)、肿瘤坏死因子(TNF-α)和白介素(IL-10)的上调表达,同时酶联免疫吸附测定结果证明Pm Toll9可促进TNF-α蛋白表达水平显著上调。斑节对虾体内细菌刺激实验结果显示无乳链球菌(Streptococcus agalactiae)可激活Pm Toll9在肝胰腺、肠、淋巴和鳃中的表达,哈维氏弧菌(Vibrio harveyi)可显著抑制Pm Toll9在肝胰腺中的表达。提示无乳链球菌可通过Pm Toll9激活Toll信号通路,引起机体免疫防御反应,而哈维氏弧菌对此过程具有一定的抑制作用。 相似文献
2.
采用生物信息学方法对斑点叉尾
3.
核转录因子NF-kB(Nuclear factor kappa B)对机体的免疫反应、炎症反应等起重要的调控作用。为了获得NF-kB基因启动子的活性报告基因,进一步研究原始脊椎动物免疫应答的机制,我们根据此前克隆得到的东北七鳃鳗(Lethenteron morii)NF-kB基因cds序列,以与东北七鳃鳗高度同源的日本七鳃鳗(Lampetra japonica)同源基因 5’上游启动子特征序列为模板设计引物,克隆获得了东北七鳃鳗NF-kB启动子序列,该序列全长3169 bp,提交至NCBI Genbank获得登录号MN368861。经AliBaba2.1软件预测该序列包含CREB (cgtgacttca)、Oct-1 (aatatgaatt)、GATA-1 (gaacctatct)、AP-1 (ggttgagtca)、NF-kappa B (ctttcctgtt)、IRF-1 (tttcctgttc)、Sp-1 (tgtgaggggt)、c-Jun (acgtgacttc)和c-Fos (ggttgagtca)等多个转录因子结合位点,并包含TATA box转录核心元件。通过MethPrimer软件预测在序列1207-1402 bp处存在CG含量大于50%,长196 bp的CpG岛。利用双酶切技术构建了pGL3-NF-kB-pro重组质粒,并将其转染至人胚肾细胞系（Human embryonal kidney, HEK293T）和鲤鱼上皮瘤细胞系（Epithelioma papulosum cyprinid, EPC）中。双荧光素酶报告基因系统检测结果显示该片段序列在哺乳动物细胞系和鱼类细胞系中均具有启动子活性,同时在HEK293T中经过Toll样受体（Toll-like receptors,TLR）的配体LPS刺激后启动子活性显著上升。本研究成功构建了东北七鳃鳗NF-kB启动子报告基因,并在不同细胞系中证实了其活性。LPS刺激后的检测结果揭示东北七鳃鳗NF-kB参与了TLR信号通路的免疫应答。东北七鳃鳗NF-kB报告基因的构建为探究原始脊椎动物免疫系统信号传导机制奠定了基础。 相似文献
4.
5.
Paul V. Zimba Al Camus JoAnn M. Burkholder 《Aquaculture (Amsterdam, Netherlands)》2006,261(3):1048-1055
Various freshwater and marine algal toxins are known to affect plants, fishes, mammals, and invertebrates. During recent mortality events in Texas white shrimp aquaculture ponds, water and shrimp tissue samples were analyzed for cyanobacterial toxins and found to contain microcystin-LR. Cyanoprokaryota dominated the phytoplankton assemblage in water from the affected pond, particularly Microcystis aeruginosa and Anabaena sp. Water samples from the affected pond also contained high levels of microcystin-LR (45 μg/l), whereas adjacent ponds had a diatom-green algal assemblage and no measurable toxin. Unialgal isolates of M. aeruginosa from the affected pond produced microcystin-LR. Free microcystin-LR concentrations in dead shrimp hepatopancreas determined by HPLC were 55 μg/g total shrimp weight, whereas shrimp hepatopancreas from the adjacent pond without shrimp mortalities had no measurable toxin. Muscle toxin concentration was below 0.1 μg/g. 相似文献
6.
7.
Catfish farms located in the south-eastern USA using brackish (3–5 g NaCl L−1 ) well water experience sporadic fish kills sometimes with high mortality. An investigation of three catastrophic losses occurring in this region identified no involvement of infectious diseases or traditional water quality problems, including oxygen, ammonia or nitrite. The high mortality and time course of the problem was indicative of exposure to a toxin. Attempts by other workers to explain the cause of this unique syndrome (high chloride associated toxicosis of catfish, HCTC), suggested that the losses might be because of microcystin-producing blooms of Microcystis aeruginosa , but our investigations failed to support this conclusion . We found that (1) the liver histology of catfish experimentally exposed to pure microcystin-LR is very different from that of catfish sampled during outbreaks of HCTC; (2) measurements of microcystin-LR concentrations in the three cases were far lower than the concentration required to kill catfish by experimental immersion; (3) the HCTC toxin appears to have a short half-life, whereas microcystin-LR does not; (4) experimental gavage of catfish with massive amounts of microcystin-LR does not cause the acute mortality typical of HCTC; (5) outbreaks of HCTC appear to be associated with heavy blooms of Anacystis marina , a halophytic cyanobacteria, not with blooms of M. aeruginosa . 相似文献
8.
9.
The second prolactin receptor in Nile tilapia (Oreochromis niloticus): molecular characterization, tissue distribution and gene expression 总被引：1，自引：0，他引：1
Yong Zhang Zijie Long Yangyuan Li Shibai Yi Yu Shi Xilan Ma Weiren Huang Danqi Lu Pei Zhu Xiaochun Liu Zining Meng Xigui Huang Christopher H. K. Cheng Haoran Lin 《Fish physiology and biochemistry》2010,36(2):283-295
10.
This study established and characterized a new cell line (MAF) from the fin of blunt snout bream (Megalobrama amblycephala), a freshwater fish cultivated in China. MAF cells proliferated well in medium 199 supplemented with 10 % fetal bovine serum at 28 °C and have been subcultured more than 95 times in almost a year. MAF cells were revived at 90–95 % viability after 3–6 months of storage in liquid nitrogen. Karyotyping indicated that the modal chromosome number of MAF cells was 48. The MAF cell line consisted predominantly of fibroblastic and epithelial-like cells from M. amblycephala, which was confirmed by immunofluorescence and mitochondrial 12s rRNA sequencing. Viral susceptibility tests showed that MAF cells were susceptible to infection by snakehead rhabdovirus, spring viremia carp virus, and channel catfish virus, which was demonstrated by the presence of cytopathic effect, high viral titers, and PCR products. Bacterial cytotoxicity studies showed that extracellular products from Aeromonas hydrophila were toxic to MAF cells. Cu2+ was also cytotoxic to MAF cells, and the 24-h IC50 value was 144.48 μmol/l. When MAF cells were transfected with pEGFP-N1 plasmid, bright fluorescent signals were observed, and the transfection efficiency reached up to 5 %. These results suggest that the MAF cell line may provide a valuable tool for studying virus pathogenesis, as well as cytotoxicity testing and genetic manipulation studies. 相似文献
11.
Flavia Bieczynski Virginia A. Bianchi Carlos M. Luquet 《Fish physiology and biochemistry》2013,39(5):1309-1321
We studied accumulation and biochemical effects of microcystin-LR (MCLR) in Odontesthes hatcheri after dietary administration of the cyanobacteria Microcystis aeruginosa (1.3 μg MCLR/g body mass, incorporated in standard fish food). After 12 h, MCLR content in liver did not differ between fish fed with crushed or intact cells, demonstrating O. hatcheri’s capacity to digest cyanobacteria and absorb MCLR. In the second experiment, fish received toxic cells, non-toxic cells, or control food; MCLR accumulation was monitored for 48 h. Protein phosphatase 1 (PP1), catalase (CAT), glutathione-S-transferase (GST) activities, and lipid peroxidation (as MDA) were measured in liver and intestine. Methanol-extractable MCLR was determined by PP1 inhibition assay (PPIA); extractable and protein-bound MCLR were measured by Lemieux oxidation-gas chromatography/mass spectrometry (GC/MS). MCLR accumulated rapidly up to 22.9 and 9.4 μg MCLR/g in intestine and liver, respectively, followed by a decreasing tendency. Protein-bound MCLR represented 66 to ca. 100 % of total MCLR in both tissues. PP1 activity remained unchanged in intestine but was increased in liver of MCLR treated fish.CAT and GST activities and MDA content were significantly increased by MCLR only in liver. We conclude that O. hatcheri is able to digest cyanobacteria, accumulating MCLR mostly bound to proteins. Our data suggest that this freshwater fish can be adversely affected by cyanobacterial blooms. However, the rapid decrease of the detectable MCLR in both tissues could imply that sublethal toxin accumulation is rapidly reversed. 相似文献
12.
13.
14.
Yudong Jia Ai Sun Zhen Meng Baoliang Liu Jilin Lei 《Fish physiology and biochemistry》2016,42(1):179-191
Molecular cloning, characterization, and functional analysis of follicle-stimulating hormone receptor (FSHR) in female turbot (Scophthalmus maximus) were evaluated. Results showed that the full-length FSHR cDNA was 3824 bp long and contained a 2202 bp open reading frame that encoded a mature protein of 733 amino acids (aa) and a signal peptide of 18 aa. Multiple sequence analyses showed that turbot FSHR has high homology with the corresponding genes of other teleosts and significant homology with that of Hippoglossus hippoglossus. Turbot FSHR has the typical structural architecture of glycoprotein hormone receptors consisting of a large N-terminal extracellular domain, seven transmembrane domains and short C-terminal intracellular domain. FSHR mRNA was found to be abundant in the ovaries, but deficient in eyes, intestine, brain, muscle, gills, spleen, stomach, heart and kidney. Furthermore, FSHR mRNA was found to increase gradually from pre-vitellogenesis to migratory nucleus stages, with the highest values observed during the late vitellogenesis stage of the reproductive cycle. However, FSHR mRNA was found to decrease dramatically during the atresia stage. Meanwhile, functional analysis with HEK293T cells continual expressing FSHR demonstrated that FSHR was specifically stimulated by ovine FSH, but not ovine LH. These results indicate that turbot FSHR is mainly involved in the stimulation of vitellogenesis, regulation of oocyte maturation as well as promotion of ovarian development via specific ligand binding. These findings open doors to further investigation of physiological functions of FSHR, which will be valuable for fish reproduction and broodstock management. 相似文献
15.
细胞周期蛋白B（Cyclin B）是影响卵母细胞发育成熟的重要基因。文章构建了PmCyB慢病毒表达载体,并和慢病毒包装复合物在293T细胞中包装成慢病毒颗粒。利用包装好的慢病毒颗粒,感染干扰了Cyclin B1基因的Hela细胞,研究了PmCyB对Hela细胞增殖的影响。结果表明,通过siRNA干扰细胞自身的Cyclin B1后,转染对照组病毒液的Hela细胞增殖减缓;同时,干扰后转入含有PmCyB重组慢病毒表达载体,Hela细胞增殖速度明显要高于转染对照组病毒液的Hela细胞的增殖速度。这一结果表明,PmCyB基因的过表达能补偿Cyclin B1缺失导致的细胞增殖减缓的现象,说明PmCyB基因具有细胞周期调控功能,是细胞增殖与分化的重要调控基因,且功能可能与人类的Cyclin B1相似。人类的Cyclin B1是卵母细胞发育成熟的重要基因。由此可以推断,PmCyB基因可能对斑节对虾（Penaeus monodon）卵巢发育成熟起着重要作用。 相似文献
16.
为更好地开展鳜基因功能研究和药物筛选工作,提高基因转染效率,本研究以鳜囊胚期胚胎为材料,采用含有20%胎牛血清的DMEM培养基进行培养,建立了生长稳定的鳜胚胎细胞系MFE。在此基础上,采用绿色荧光蛋白(GFP)作为标记物,在HEK293T细胞中体外包装逆转录病毒,再感染MFE细胞系。MTT法分析表明传代后细胞培养96 h内,细胞生长率变化也经历增殖、降低到达稳定期。而且MFE细胞能稳定表达GFP基因,感染效率为20%±5%,而脂质体转染效率为3%±2%。可见包装病毒感染细胞不仅能获得稳转细胞系,效率也远高于脂质体瞬时转染。荧光定量PCR分析表明,MFE细胞系能表达Irf1、Irf3和Irf7基因,Irf1基因表达量最高。MFE细胞系受到poly I:C刺激后,Irf1、Irf3和Irf7的表达量分别升高3.5,2.3和2.1倍。因此,MFE细胞通过病毒感染可以获得较高的转染效率,该细胞可作为鳜免疫相关基因功能研究的工具。 相似文献
17.
Marine-derived actinomycetes (87 strains isolated from seaweed and 98 strains isolated from marine sediment) were screened for antimicrobial activity against bacterial fish pathogens. The most potent active strain (PK288-21) isolated from the rhizosphere of the marine seaweed Undaria pinnatifida was identified as Streptomyces atrovirens by 16S rDNA sequence analysis. Two active compounds were isolated from a culture extract of strain PK288-21 by silica-gel flash chromatography and high-performance liquid chromatography. The structures of the two compounds were identified as 2-hydroxy-5-(3-methylbut-2-enyl)benzaldehyde (B1) and 2-hepta-1,5-dienyl-3,6-dihydroxy-5-(3-methylbut-2-enyl) benzaldehyde (B2) by nuclear magnetic resonance and high-resolution fast atomic bombardment mass spectroscopy. The antimicrobial activities of the two compounds were tested against bacterial fish pathogens and expressed in terms of the minimum inhibitory concentration (MIC). Metabolite production was found to be optimized in A1BFe medium following the screening of eight different media. The two compounds killed Edwardsiella tarda after 12?h and Streptococcus iniae after 16?h at the MIC. Compound B1, 2-hydroxy-5-(3-methylbut-2-enyl)benzaldehyde, is a new benzaldehyde derivative, and this is the first time that either of these compounds have been reported in the genus Streptomyces. 相似文献
18.
为获得尼罗罗非鱼Siglecs like融合蛋白,开展相关Siglecs like蛋白的功能研究,深入了解罗非鱼与无乳链球菌相互作用机制。本研究利用前期构建的3个含Siglecs like ORF的克隆质粒,PCR扩增获得Siglec-1、Siglec-4b和Siglec-14 like的膜外段序列,插入真核表达载体pc DNA3.1(+)h Ig G1 Fc中,双酶切、测序鉴定后转染COS-7细胞。q PCR、Western-blot对目的蛋白的表达进行检测;亲和层析法纯化融合蛋白,SDS-PAGE电泳检测纯化效率。ELISA检测融合蛋白与GBS的结合活性。测序结果显示,成功构建3个融合蛋白真核表达载体pc DNA3.1(+)h Ig G1 Fc-Siglecs like/Ex。检测结果显示,转染细胞中3个Siglecs/Ex-Fc融合蛋白在mRNA和蛋白水平都有高效表达,且过柱后的融合蛋白具有较高纯度;3个融合蛋白与罗非鱼源GBS的结合活性较对照组均有显著差异。研究表明,利用真核表达系统成功制备了具有较高纯度的尼罗罗非鱼3种Siglecs融合蛋白,且均有与GBS的结合活性。 相似文献
19.
Ganesan Nagarajan Adimoolam Aruna Ching-Fong Chang 《Fish physiology and biochemistry》2013,39(1):95-101
Despite neurosteroidogenic enzymes are playing important roles in the regulation of brain development and function, the potential link between brain and gonad by the action of steroid hormones during gonadal sex differentiation is still not clear in teleosts. In this mini-review, we summarized our understanding on the early brain development related to the synthesis of neurosteroids and receptor signaling during gonadal sex differentiation in protogynous orange-spotted grouper, Epinephelus coioides (functional females for the first 6 years of life and start to sex change around the age of 7 years) and protandrous black porgy (functional males for the first 2 years of life but begin to change sex during the third year). We found a similar profile in the increased expression of brain aromatase gene (aromatatse B or cyp19a1b), aromatase activity, estradiol (E2), and estrogen signaling in the brain of both grouper and black porgy fish during gonadal sex differentiation. In contrast to mammals, teleost fish Cyp19a1b expressed in a unique cell type, a radial glial cell, which is acted as progenitors in the brain of developing and adult fish. In agreement with these pioneer studies, we demonstrated that the grouper cyp19a1b/Cyp19a1b was expressed in radial glial cells. Further, in vivo data in the grouper brain showed that exogenous E2 upregulated Cyp19a1b immunoreactivity (ir) in radial glial cells. These data suggest the possible roles of Cyp19a1b and E2 in early brain development which is presumably related to gonadal sex differentiation. 相似文献
20.
Noriyoshi Sakai Minoru Tanaka Mika Takahashi Shinji Adachi Yoshitaka Nagahama 《Fish physiology and biochemistry》1993,11(1-6):273-279
A distinct shift in steroidogenesis from testosterone to 17α-hydroxyprogesterone occurs in the salmonid ovarian thecal cell layers immediately prior to oocyte maturation, and is a prerequisite for the production of 17α,20β-dihydroxy-4-pregnen-3-one (maturation-inducing hormone of salmonid fishes) by granulosa cells during oocyte maturation. 17α-Hydroxylase/17,20-lyase cytochrome P-450 (P-45017α) and 3β-hydroxysteroid dehydrogenase/Δ5?4-isomerase (3β-HSD) are the two major steroidogenic enzymes involved in the production of 17α-hydroxyprogesterone and testosterone. Using mammalian cDNA probes, we isolated and characterized full-length cDNAs encoding these two enzymes from a rainbow trout (Oncorhynchus mykiss) ovarian thecal cell cDNA library. The cloning of 2.4-kilobase cDNA encoding P-45017α and transient expression of this clone in nonsteroidogenic monkey kidney tumor COS-1 cells have recently been reported (Sakai et al. 1992). We have isolated a 1.4-kilobase cDNA which is hybridized to the mammalian 3β-HSD cDNAs. Expression of this cDNA in COS-1 cells led to the production of an enzyme which is capable of converting dehydroepiandrosterone to androstenedione. In this study, enzymatic activities and expression of rainbow trout ovarian P-45017α and 3β-HSD are discussed in relation of the steroidogenic shift occurring in the ovarian follicle layers. 相似文献