褶纹冠蚌过氧化物还原酶6基因的克隆及原核表达

过氧化物还原酶6具有谷胱甘肽过氧化物酶和磷脂酶A2的双重活性,在机体抗氧化保护及肺表面活性物质代谢中具有重要的作用。研究克隆和分析了褶纹冠蚌Prx6(CpPrx6)基因的cDNA序列特征。结果表明CpPrx6基因的cDNA全长1617 bp;其中5′端非翻译区为71 bp,3′端非翻译区为889 bp,开放阅读框为657bp,可以编码218个氨基酸。CpPrx6氨基酸序列与其他已知贝类Prx6的同源性为70%—72%。CpPrx6蛋白含有1-Cys型Prx共有的保守催化中心"PVCTTE"和脂肪酶基序"GKSWA",三级结构中包含6个α螺旋、12个β折叠,催化中心位于第5个α螺旋内。CpPrx6基因在褶纹冠蚌血细胞、外套膜、闭壳肌、肝胰腺、鳃等组织中均有表达,其中鳃的表达量最大。嗜水气单胞菌刺激后6h和12h时CpPrx6在肝胰腺中的表达量明显增加(6h,P<0.05;12h,P<0.01),在血细胞和鳃组织中12h的表达量增加,24h时恢复到正常水平。将CpPrx6基因亚克隆到pET-32a(+)质粒中构建了重组质粒,SDS-PAGE分析发现重组质粒在大肠杆菌DE3中获得了表达。     

池蝶蚌组织蛋白酶L基因的组织表达及免疫应激分析

应用cDNA文库筛查及同源片段克隆拼接技术,克隆了池蝶蚌组织蛋白酶L(Hs-CtsL)cDNA基因全长序列(GenBank注册号为JN604558)。其cDNA全长1152 bp,5′-非翻译区(Untranslated Region,UTR)长1 bp,3′-UTR长149 bp包括1个多聚腺苷信号AATAAA和Poly(A)尾巴,开放阅读框(Open reading frame ORF)为1002 bp,编码333个氨基酸组成的多肽链。其分子量约37.7 kD,理论等电点为7.16,包含信号肽、前体域和成熟域。系统进化分析显示,Hs-CtsL同无脊椎动物组织蛋白酶L聚为一支,且同三角帆蚌亲缘关系最近,其次为褶纹冠蚌。组织表达分析结果显示,池蝶蚌组织蛋白酶L在肠、鳃、性腺、外套膜、斧足、闭壳肌、血细胞、肝胰腺、肾和心脏均有表达,其中血细胞中表达量最高。应激实验表明,经嗜水气单胞菌刺激后,Hs-CtsL在血细胞、鳃、肝胰腺和外套膜中的表达量显著上调。其中在肝胰腺中刺激后6h表达量到达峰值,在血细胞、鳃和外套膜中的表达模式近似,表现为一个波动变化,在4h、12h和48h被上调。结果暗示着Hs-CtsL除参与了池蝶蚌血细胞的先天性免疫防御以外,还参与了其消化腺免疫器官的免疫应答反应。     

三角帆蚌alpha-2巨球蛋白活性测定及不同组织的表达

alpha-2巨球蛋白(alpha-2 Macroglobulin,α2M)是一种蛋白酶结合蛋白,是重要的天然免疫因子。利用α2M的作用原理,用三角帆蚌血浆保护胰蛋白酶,后用大豆蛋白酶抑制剂抑制未被保护的胰蛋白酶。利用Na-benzoyl-DL-arg-nine-p-nitroanilide(BAPNA)作为酶底物检测被保护的蛋白酶活性的方法,首次证明了三角帆蚌体内α2M的存在。在此基础上,克隆了α2M基因保守区受体结合区片段,进一步证明了α2M在三角帆蚌体内的存在。同时,还进行了α2M不同组织的表达检测,结果显示,α2M基因在血细胞中有表达,而在外套膜、闭壳肌、肠和性腺中没有表达。     

淡水育珠蚌褶纹冠蚌血细胞的初步研究

国外关于海产双壳贝血细胞形态与功能的研究虽然较多,但对于淡水育珠蚌褶纹冠蚌的血细胞却无研究报道,国内关于软体动物血细胞的研究甚少,对于育珠蚌血细胞只笼统地称之为游走细胞,颗粒细胞等,对它们的种类和形态无详细的描述。为此作者对褶纹冠蚌的血细胞进行了形态学的初步观察,以期对今后的研究工作提供参考。     

中华鲟养殖池背角无齿蚌和鲢鳙鱼生态修复效果比较

2009年5月31日-6月20日在广东省大沙河水库利用微型生态系统比较不同放养密度的褶纹冠蚌(Cristaria plicata)和背角无齿蚌(Anodonta woodiana)对水体氮、磷及浮游植物的影响,探讨两种蚌在控制南亚热带水库富营养化水体藻类水华上的可行性。实验结果表明,在褶纹冠蚌和背角无齿蚌处理组中,总氮、总磷和叶绿素a浓度显著增加,而铵氮的浓度显著下降;褶纹冠蚌和背角无齿蚌导致了浮游植物群落组成结构的改变和数量的增加,实验过程中绿藻所占的比例迅速上升。两种蚌之间没有显著的差异,只是在不同的作用强度下,时间上的响应不同。综合实验结果,褶纹冠蚌和背角无齿蚌难以有效地运用于我国华南地区水库的水质改善与富营养化控制。     

褶纹冠蚌和背角无齿蚌对水体营养盐和浮游植物的影响

2009年5月31日-6月20日在广东省大沙河水库利用微型生态系统比较不同放养密度的褶纹冠蚌(Cristaria plicata)和背角无齿蚌(Anodonta woodiana)对水体氮、磷及浮游植物的影响,探讨两种蚌在控制南亚热带水库富营养化水体藻类水华上的可行性。实验结果表明,在褶纹冠蚌和背角无齿蚌处理组中,总氮、总磷和叶绿素a浓度显著增加,而铵氮的浓度显著下降;褶纹冠蚌和背角无齿蚌导致了浮游植物群落组成结构的改变和数量的增加,实验过程中绿藻所占的比例迅速上升。两种蚌之间没有显著的差异,只是在不同的作用强度下,时间上的响应不同。综合实验结果,褶纹冠蚌和背角无齿蚌难以有效地运用于我国华南地区水库的水质改善与富营养化控制。     

氨对褶纹冠蚌钩介幼虫及稚蚌的急性毒性

为探究氨对蚌类早期生活史阶段的毒性效应,研究分别以褶纹冠蚌(Cristaria plicata)的钩介幼虫及稚蚌为实验对象开展了24h和48h的急性毒性实验。结果表明:氨对褶纹冠蚌钩介幼虫的24h半致死浓度(LC₅₀)为0.63 mg NH₃-N/L (NH₃,分子氨)和78 mg TAN_7.0,20℃/L (TAN_7.0,20℃,pH 7.0水温20℃时即标准化的总氨氮);氨对褶纹冠蚌稚蚌的48h LC₅₀为0.60 mg NH₃-N/L和104 mg TAN_7.0,20℃/L;上述阈值高于已有研究中氨对其他淡水蚌类的毒性阈值;褶纹冠蚌钩介幼虫对氨氮的耐受性低于稚蚌,二者均低于幼蚌,因此褶纹冠蚌更早期生活史阶段对氨的耐受性更低。相关研究结果可完善氨对蚌类毒性的理解,为水体氮管理策略的制定和蚌类保护提供一定科学依据。     

池蝶蚌和三角帆蚌谷胱甘肽S转移酶基因表达特征

采用RT-PCR方法克隆获得池蝶蚌(Hyriopsis schlegerlii)和三角帆蚌(H.cumingii)GST片段,两种蚌的GST核苷酸序列相同,推导其编码92个氨基酸;经与不同物种进行氨基酸序列比对,显示其与软体动物、两栖动物和高等哺乳动物的GST具有高度同源性。RT-PCR半定量分析表明,GST在两种蚌的肝、闭壳肌等11种组织中除肠以外均有表达,肝中表达量较大;注射嗜水气单胞菌(Aeromonas hydrophila)后,三角帆蚌血细胞中GST表达量先降低后升高,而池蝶蚌血细胞中GST表达量先升高后降低,且在感染3h、6h、9h和24h时表达量约为三角帆蚌的2倍。     

几种淡水蚌外套膜蛋白质氨基酸组成及部分元素分析

测定了三角帆蚌、圆背角无齿蚌、褶纹冠蚌、背视丽蚌外套膜的粗蛋白含量 ,分析了外套膜干粉的氨基酸组成 ,并采用原子吸收光谱法和等离子吸收光谱法测定三角帆蚌、圆背角无齿蚌、褶纹冠蚌外套膜的 12种元素含量。结果表明 :干燥外套膜蛋白质含量为 :三角帆蚌 4 1 34% ,圆背角无齿蚌2 2 0 3% ,褶纹冠蚌 32 14 % ,背视丽蚌 7 78%。这四种淡水蚌外套膜均含有 17种氨基酸。被测的 12种元素中 ,Cu元素含量最高 ,其次是Fe ,Mn ,Mg ,Ca ,Zn ,Cr,而Ni,Co ,Pb ,Hg等元素含量较低。这四种淡水蚌外套膜是一类优质的动物蛋白质资源。     

人工采集褶纹冠蚌钩介蚴虫的点滴经验

我们从1972年开始人工繁殖褶纹冠蚌,通过两年的工作认识到褶纹冠蚌的人工繁殖较三角帆蚌要容易得多。但在采集钩介蚴虫的过程中,往往遇到的困难,就是成熟的褶纹冠蚌母体在拿进室内放入盛水的容器中,排蚴的速度极其缓慢,有的母蚌由于长期不排蚴而引起外鳃瓣中钩介蚴虫的萎缩、退化现象,因此采取杀蚌取蚴的方法。我场在1972年初也曾采用此法,后来我们遵照毛主席关于“要认真总结经验”的教导反复实践;不断总结,发现同日成熟的母蚌,当所处的环境条     

Immunodetection of hemocytes, peneidins and α2-macroglobulin in the lymphoid organ of white spot syndrome virus infected shrimp

Viral diseases restrict the development of the world shrimp industry and there are few studies on cell response to the presence of viral infections. We performed immunohistochemistry assays to characterize hemocytes subpopulations involved in the immune process occurring in the LO of Litopenaeus vannamei shrimp. Tissue sections of animals that increased their LO spheroids and hemocytes infiltration after WSSV induced infection, were used. Three MABs namely, 40E10 (recognizing small granule hemocytes), 40E2 (recognizing large granule hemocytes), and 41B12, which recognize α(2)-macroglobulin were used. Additionally one polyclonal antibody was used against the penaeidins antimicrobial peptides, and to detect WSSV a commercial immunohistochemistry kit (DiagXotics) was used. Numerous small granule hemocytes were detected in the stromal matrix of LO tubules, whereas large granule hemocytes were less numerous and located mainly in hemal sinuses. The exocytosis of two molecules, which have been related to the phagocytosis process, i.e. penaeidins, and α(2)-macroglobulin, was detected in the external stromal matrix and the outer tubule walls. α(2) -macroglobulin inhibits phenoloxidase activity and its strong release in LO tissue may explain the absence of melanization in the immune processes occurring in it. The immunolabeling of vesicles within the LO spheroids with MABs 41B12 40E10 and antipenaedin antibody suggests that LOS are formed by phagocytic cells derived from small granule and hyaline hemocytes, with a possible role of peneidins and α(2)-macroglobulin acting as opsonines.     

Immune role of MrNFκBI-α, an IκB family member characterized in prawn M. rosenbergii

NF kappa B inhibitor alpha (MrNFκBI-α) was sequenced from a freshwater prawn Macrobrachium rosenbergii. The MrNFκBI-α protein contains a long ankyrin repeat region circular domain between 193 and 413 along with its 6 repeats (ankyrin repeat 1,2,3,4,5 and 6). An IκB degradation motif and a putative PEST motif is present at 37-64 and 418-471 of the N- and C-terminal regions of MrNFκBI-α respectively. The gene expressions of MrNFκBI-α in healthy and infectious hematopoietic and hypodermal necrosis virus (IHHNV), poly I:C, Aeromonas hydrophila and Enterococcus faecium injected M.?rosenbergii were examined using quantitative real time PCR. The MrNFκBI-α is expressed in all the tissues taken for examination and the highest is observed in hemocytes. The MrNFκBI-α gene expression is strongly up-regulated in hemocytes of prawn after IHHNV, poly I:C, A.?hydrophila and E.?faecium infection. This result indicates an important role of MrNFκBI-α in M.?rosenbergii immune system. This, however, remains to be verified by further studies.     

Differentially enhanced gene expression in hemocytes from Macrobrachium rosenbergii challenged in vivo with lipopolysaccharide

In order to better understand the immune response in prawns after treatment with the immunostimulant lipopolysaccharide (LPS), in this study, the differential gene expression of the hemocytes from LPS-injected versus non-injected prawns (Macrobrachium rosenbergii) were isolated and identified using suppression subtractive hybridization (SSH). The hemocytes were extracted after treatment for 1, 6, and 12 h. The upregulated genes (i.e., where gene expression was elevated) were identified and could be divided into four classes on the basis of physiological function: genes concerning defense-related molecules, genes involved in energy-production (respiration), genes related to protein synthesis and folding, and genes with unknown function. The time-course for gene expression indicated that, except for expression of the gene anti-microbial peptide (amp), which was increased at 12 h after LPS treatment, the expression of the other two immune-related genes was much earlier (at 1 h), including alpha-2-macroglobulin (α2-M) and Mas-like protein (mas). These results suggest that in the early phase of LPS stimulation some immune reactions regulated by α-2M and Mas may be induced, such as the activation of prophenoloxidase activating system, opsonization, and anti-microbial activity. In addition, six unigenes with unproven function were particularly interesting and worthy of further study because their expression in LPS-treated hemocytes was clearly enhanced.     

日本沼虾两种抗脂多糖因子的特性研究

为了研究抗脂多糖因子ALFs在日本沼虾先天性免疫中的功能作用, 研究从日本沼虾中克隆了2种抗脂多糖因子MnALF1、MnALF2。MnALF1 cDNA 全长1008 bp, 编码121个氨基酸; MnALF2 cDNA 全长836 bp, 编码124个氨基酸。这2种氨基酸均包含有一个信号肽序列和一个LPS结合位点, 并且在结合位点的两端(N-端和C-端)都有2个保守的半胱氨酸残基。这2种MnALFs与之前发现的甲壳动物的ALFs是非常相似的。qRT-PCR结果显示MnALFs在所有被检测的组织中均有表达。其中MnALF1主要在心脏和小肠内表达, 而MnALF2则主要在血细胞和肝胰脏中表达。在用嗜水气单胞菌刺激之后发现2种MnALFs在心脏、小肠、血细胞、肝胰脏中都呈现出明显的时间依赖表达模式(MnALF1在刺激之后呈现出先减少后增加的趋势, 之后分别在不同组织的不同时间点达到最大值; 然而, 对于MnALF2, 在心脏和小肠中先减少后增加, 在血细胞和肝胰脏中呈现出先增加后减少, 最后都在24h达到最大值)。结果提示这2种MnALF具有不同的组织特异性, 并且在细菌侵染的免疫防御中起着重要的保护作用。     

α2巨球蛋白的应用研究进展

α2巨球蛋白(α2-macroglobulin,α2M)是血浆中含量丰富的大分子糖蛋白,是一种广谱的蛋白酶抑制剂,主要通过调节内源或外源性蛋白酶广泛参与许多重要的生理病理过程。α2M还通过与细胞因子、生长因子和激素等分子结合调节它们在体内的分布、半衰期和生物学活性。研究发现α2M具有广泛的应用前景:α2M对辐射损伤、脓毒症、阿尔茨海默病等疾病具有很好的保护作用。此外,α2M还可以作为药物传递的载体和疫苗佐剂,并且可以作为许多疾病诊断和预后的生物标志物。本文主要综述α2M在应用方面的新进展。     

Cloning, distribution and primary immune characteristics of amphioxus alpha-2 macroglobulin

Alpha-2 macroglobulin (α(2)M), a broad-spectrum protease inhibitor, exists widely in vertebrates and invertebrates, but little information is available to date regarding α(2)M in amphioxus, an animal bridging from invertebrates to vertebrates. Here we first show that the full α(2)M cDNA of Branchiostoma japonicum (Bjα(2)m) contained 5545 bp with an open reading frame of 4593 bp encoding signal sequence of 16 amino acid residues and a mature protein of 1514 residues. The calculated molecular mass and pI of mature Bjα(2)M were 164.2 kDa and 4.6 respectively. Bjα(2)m was mainly expressed in the hepatic caecum and hind-gut in a tissue-specific manner, contrasting to the primary expression of α(2)M in vertebrate liver. Following challenge with lipopolysaccharide (LPS), Bjα(2)m expression was significantly up-regulated (7-folds) at 8 h and then declined to the base line at 16 h. Taken together, it is suggested that Bjα(2)M is an immune-relevant molecule possibly involved in the acute phase response via the digestive organs.     

大鼠肝癌中α_2-巨球蛋白等几种分泌性蛋白基因表达的变化

用Northern blot方法对二乙基亚硝胺所诱发的大鼠肝癌中内源性蛋白酶抑制因子α_2-巨球蛋白(α_2-M)、非特异性免疫抑制剂α_1-酸性糖蛋白(α_1-AGP)及雄性激素正调控的α-2u球蛋白(α-2u)三种分泌性蛋白基因表达情况进行了分析。结果表明在大部分(14/16)肝癌样品中α_2-M RNA水平显著降低;而α_1-AGP RNA水平显著高于正常对照水平;α-2u RNA水平明显下降,但在某些雄性大鼠肝癌样品中该基因却有一定程度的表达。这些结果说明,一些肿瘤宿主血浆中α_2-M水平的显著下降及α_1-AGP水平的明显升高分别是由于基因表达活性的下降及升高所致。α-2u基因表达的异常提示,在癌变过程中机体的内分泌功能发生了某些变化。     

Analysis of four circulating complexes of insulin-like growth factor binding proteins in human blood during aging

The primary role of insulin-like growth factor binding proteins (IGFBPs) is to regulate availability of IGFs for interacting with receptors, but IGFBPs perform IGF-independent actions as well. The availability and activity of IGFBPs in the circulation is influenced primarily by their concentration and structural modifications, but possibly also by interaction with major plasma proteins such as transferrin, alpha-2-macroglobulin (α2M), and fibrinogen. Four types of circulating IGFBP complexes were examined in this study by immuno- and ligand-binding assays in adults of different age. The amounts of IGFBP-3/transferrin and IGFBP-1/fibrinogen complexes were similar in middle- and old-aged persons, whereas the amounts of IGFBP-1 (or -2)/α2M monomer complexes were lower in the old-aged group and negatively correlated with total IGFBP-1 (or -2) amounts in blood. In contrast to IGFBP-1, IGFBP-2 was present in significantly greater quantities in complexes with α2M dimer than α2M monomer in older individuals. IGFBP complexes did not bind ¹²⁵I-labeled IGF-I in amounts detectable by ligand blotting. According to the results of this study, the quantities of IGFBP-1 and IGFBP-2, which interact with α2M, are age-dependent and, in the case of complexes with α2M monomer, they are negatively correlated with the total circulating levels of these two IGFBPs.