草鱼RAGs的克隆及不同发育阶段的表达分析

草鱼(Ctenopharyngodon idellus)在1、2龄阶段的抗病能力很差,但到3龄阶段后义具有了很强的抗病能力,提示草鱼的免疫系统可能要到3龄发育阶段才会完善.重组激活基因(Recombination activating genes,RAGs)编码的蛋白质专一性介导淋巴细胞抗原识别受体基因的V(D)J重组,在脊椎动物淋巴细胞正常发育成熟过程中起关键作用.为了进一步研究草鱼免疫系统建立和完善的发育遗传学因素,通过PCR方法克隆了草鱼重组激活基因rag1和rag2,分析了它们在不同发育阶段的表达.草鱼rag1基因从起始密码到终止密码总长4188 bp,由三个外显子和两个内含子组成,其开放阅读框长3192 bp,编码1063个氨基酸.草鱼rag2基因从起始密码到终止密码总长1593bp,没有内含子,编码530个氨基酸.氨基酸序列比对分析表明,预测的草鱼RAG1和RAG2蛋白的功能区域与其他脊椎动物的相应功能区具有很高的同源性.从受精后第4天开始,RT-PCR即可检测到rag1基因的表达,在随后的幼鱼阶段rag1基因维持了高水平的表达.在1龄草鱼的头肾中仍可以检测到rag1基因的表达,但在3龄草鱼头肾中没有检测到rag1基因的表达.这些结果表明草鱼从胚胎的孵化期开始到1龄幼鱼阶段是免疫球蛋白(Ig)和T细胞受体(TCR)V(D)J重组的活跃时期,在3龄发育阶段其淋巴细胞系中V(D)J重组活动已经很少,免疫系统中Ig和TCR库的多样化过程趋于完成,其免疫系统已经比较完善.     

双峰驼卵泡发育过程中卵泡液差异蛋白质组学

为了比较双峰驼卵泡发育过程中卵泡液中差异表达的蛋白质,将卵泡按照直径分为6 类,运用双向电泳技术构建双峰驼卵泡液蛋白质二维凝胶电泳图谱,凝胶经考马斯亮蓝染色后用PDQuest 8. 0 检测差异蛋白,结果表明在6 类大小不同的卵泡中共检测到13 个差异蛋白点,这些蛋白点经LC -MS/ MS 鉴定出7 种不同的蛋白质,它们分别是:血红蛋白、toll-like 受体9、抗凝血酶、聚泛素、γ 纤维蛋白原、重组活化蛋白1 和跨膜与卷曲螺旋域3。基于这些蛋白的功能和表达模式,结合实验结果讨论了这些蛋白质在生殖中的功能,发现toll-like 受体9、聚泛素、γ 纤维蛋白原和重组活化蛋白1 可能与卵泡发育或卵母细胞的成熟有关。这些蛋白质的发现为了解双峰驼卵泡发育和卵母细胞成熟的生理机制奠定基础。     

GCRV感染存活草鱼的血清蛋白表达特性

为挖掘草鱼关键抗性分子,研究利用4D label-free定量蛋白质组学技术系统分析了团队前期获得的GCRV感染存活草鱼(候选抗性草鱼)与对照草鱼的血清蛋白表达差异。共鉴定到858个草鱼血清蛋白, 329个蛋白在两类草鱼中差异表达,其中163个蛋白在候选抗性草鱼血清中显著高表达, 166个蛋白显著低表达。差异表达蛋白的功能主要注释为体液免疫反应调节相关蛋白,显著富集到补体凝血级联、细胞黏附和铁死亡等信号通路。对差异表达的免疫相关蛋白进行分析发现,候选抗性草鱼血清中体液免疫分子MASP2、C4a、C4b、C7b、C8b、C8g、C9、CFI、C3a.1、C3a.2、C3a.3和C3a.6等补体和抗原提呈相关免疫分子MHC1UBA和IGL4V8等蛋白表达水平显著高于对照草鱼,而T细胞免疫相关分子TRAV、IGHV1-1、IGHV2-1、IGHV6-1、IGHV3-2和IGHV11-2等蛋白表达水平显著低于对照草鱼。免疫印迹检测进一步证实,以C3和IgM为代表的体液免疫分子在候选抗性草鱼血清中显著高表达,可能与草鱼抗病能力关联。研究将为高抗性草鱼的选育提供可参考的分子资源。     

不同龄期移虫发育的蜂王卵巢蛋白质组学分析（英文）

【目的】探讨移虫龄期对西方蜜蜂Apis mellifera蜂王卵巢发育的分子调控机制。【方法】运用相对和绝对定量同位素标记(iTRAQ)技术对不同龄期移虫培育的蜂王卵巢组织蛋白质进行定量分析,筛选差异表达蛋白。利用蛋白质免疫印迹法对结果进行验证。【结果】iTRAQ定量分析共鉴定到蜂王卵巢组织蛋白质二级图谱452 966个,最终获得3 642个蛋白。GO(Gene Ontology)富集结果表明,不同龄期移虫发育的蜂王卵巢组织差异表达蛋白主要富集在细胞代谢、分裂以及蛋白质合成。Pathway富集分析表明,1龄幼虫期移虫和2龄幼虫期移虫发育的蜂王卵巢组织差异表达蛋白主要富集在碳水化合物代谢、脂代谢和外源降解类群;1龄幼虫期移虫和3龄幼虫期移虫发育蜂王卵巢组织差异蛋白主要富集在有机体发育通路、核糖体通路和溶酶体代谢。与此同时,两个差异表达蛋白即储存蛋白Hexamerin110和Hexamerin70b的蛋白免疫印迹检测结果表明,随着移虫龄期的增加,Hexamerin110和Hexamerin70b的表达量均呈现降低的趋势。【结论】对不同龄期移虫的蜂王之间差异表达蛋白进行鉴定,为进一步研究蜂王生殖发育及级型分化的调控机制提供理论依据。     

蛋白质组学技术筛选和鉴定白化黑线仓鼠皮肤白化相关差异蛋白质

目的比较黑线仓鼠及其白化突变系背部皮肤蛋白表达的差异,寻找差异蛋白质,从蛋白质水平探讨白化病的发生机制。方法应用双向凝胶电泳技术分离出差异蛋白质,用质谱法分析其结构与组成,通过蛋白质数据库确定差异蛋白的功能。结果从64个表达差异蛋白斑点中发现33个显著差异的蛋白点,其中又有14个差异点匹配到了有意义的蛋白质。14个差异点共鉴定出11个差异蛋白质,这些差异蛋白质按功能可分为4类:(1)糖代谢相关蛋白;(2)运输蛋白;(3)细胞骨架蛋白;(4)其他蛋白。结论黑线仓鼠与其白化突变系背部皮肤蛋白表达存在明显差异,其中一些蛋白与白化病发生相关,并可能成为白化病致病机理研究的分子标志物和药物治疗靶向位点。     

HIV感染导致MT4细胞蛋白表达差异的初步研究

为了比较MT4细胞株感染H1V-1的IIIB株前后的蛋白质表达差异,我们分别提取MT4细胞及感染了人类免疫缺陷病毒(HrV)的MT4细胞的总蛋白质,通过双向电泳分离,使用Image Master 2D Elite 3．10图像分析软件分析获得的凝胶图谱,寻找差异点,使用质谱仪鉴定获得的差异点蛋白质。结果表明感染HIV和未感染HIV的MT4细胞有40个蛋白质点差异,HIV感染后减少的蛋白质点有12个,增多的有28个,通过质谱分析,29个蛋白质得到鉴定。其中HIV感染后下调的蛋白质有能量代谢相关蛋白、肌动蛋白相关蛋白及假想蛋白等;上调的蛋白有肌动蛋白、酶类蛋白、免疫蛋白及假想蛋白等。通过研究我们可以看出宿主细胞感染HIV病毒后有多个蛋白发生变化,可能和HIV与宿主细胞的相互作用有关。为了研究HIV感染的机制必须去除高丰度蛋白,针对特定功能的蛋白质进行具体研究。     

黄芪多糖免疫草鱼母本对子代IgM、C3和LSZ表达特性研究

为探究黄芪多糖免疫草鱼母本所产子代早期发育阶段体液免疫因子IgM、C3和LSZ的表达特性及代间传递效率。采用ELISA、RT-qPCR等方法分析了饲喂黄芪多糖草鱼母本血液及其子代早期发育阶段3种免疫因子的蛋白质活性及mRNA水平。结果显示,黄芪多糖免疫草鱼母本血液中IgM、C3和LSZ蛋白活性均显著高于对照组。在子代早期阶段中,3种免疫因子蛋白活性呈先下降后上升趋势,实验组IgM蛋白活性在各阶段均显著高于对照组,在卵子、5d和28d仔鱼中分别提高了2.2倍、1.7倍和1.8倍。实验组C3活性在卵子、24h胚胎分别提高了1.9倍和1.6倍。实验组LSZ活性在卵子、5d和28d仔鱼中分别提高了2.4倍、2.0倍和1.9倍;在卵子和受精卵时期3种免疫因子mRNA水平显著高于对照组。在24h胚胎至5d仔鱼没有检测到IgM和LSZ mRNA,而在14d后3种免疫因子的mRNA均呈上调表达,但Ig M和C3 mRNA水平与对照组无显著差异。GCRV攻毒后,实验组2月龄草鱼脾脏和头肾中IgM mRNA水平显著高于对照组。结果表明,黄芪多糖能够提高草鱼母本免疫能力并向子代垂直传递,在应对GCRV感染时发挥一定免疫保护作用。     

果梅完全花与不完全花的差异蛋白分析

应用双向电泳技术对果梅完全花与不完全花的蛋白质组分进行了比较分析。经专业分析软件（PDQuest）对电泳图谱分析表明两者的蛋白分布相似,在完全花中发现了1个特异蛋白、1个上调蛋白、21个下调蛋白,在不完全花中发现2个特异蛋白,这些蛋白差异点可能与雌蕊的败育有关。应用质谱技术对3个特异点及5个差异大的蛋白点进行分析,得到的肽段数据与蛋白质数据库比对发现其中一个蛋白（28．2kD,pI4．53）与光敏色素B有关。     

HIV感染导致MT4细胞蛋白表达差异的初步研究

为了比较MT4细胞株感染HIV-1的ⅢB株前后的蛋白质表达差异,我们分别提取MT4细胞及感染了人类免疫缺陷病毒(HIV)的MT4细胞的总蛋白质,通过双向电泳分离,使用Image Master 2D Elite 3.10图像分析软件分析获得的凝胶图谱,寻找差异点,使用质谱仪鉴定获得的差异点蛋白质.结果表明感染HIV和未感染HIV的MT4细胞有40个蛋白质点差异,HIV感染后减少的蛋白质点有12个,增多的有28个,通过质谱分析,29个蛋白质得到鉴定.其中HIV感染后下调的蛋白质有能量代谢相关蛋白、肌动蛋白相关蛋白及假想蛋白等;上调的蛋白有肌动蛋白、酶类蛋白、免疫蛋白及假想蛋白等.通过研究我们可以看出宿主细胞感染HIV病毒后有多个蛋白发生变化,可能和HIV与宿主细胞的相互作用有关.为了研究HIV感染的机制必须去除高丰度蛋白,针对特定功能的蛋白质进行具体研究.     

陆地棉‘徐州142’纤维不同发育时期蛋白质组比较分析

该研究以陆地棉‘徐州142’为材料,于开花后5、10、15、20、25、30d分别取样(棉铃)代表6个不同时期的棉纤维,利用双向电泳技术比较了棉纤维发育不同时期蛋白质组的变化,以明确不同发育阶段差异表达蛋白质及其功能与纤维发育之间的关系,为棉花产量和品质的改良提供理论依据。结果显示:(1)随着棉花的发育,棉纤维中的总蛋白含量逐渐降低,在开花后5d(5DPA)时棉花纤维中总蛋白的含量最高,达到11.7%,同时2-DE图谱中发现了15个明显的差异蛋白点;(2)运用MALDI-TOF-MS对差异蛋白质点鉴定结果发现,有5个明显的差异蛋白与棉花纤维发育相关,分别是GhSAM、GhPGK、GhCSD、GhFB和GhMDH蛋白;(3)开花后不同时期棉纤维蛋白质组的2-DE图谱表明,GhSAM、GhPGK1和GhMDH 3个蛋白在整个发育期都表达,但GhCSD从15DPA开始表达,GhFB从20DPA开始表达,而且GhSAM在15DPA时蛋白表达量最高,GhPGK1在10DPA时表达量最高,GhMDH、GhCSD和GhFB都是在30DPA时表达量最高。研究表明,棉纤维发育不同时期存在着差异蛋白,且在纤维发育不同时期蛋白的表达量也存在差异,同时这些差异蛋白参与能量代谢、碳代谢、细胞周期调控和发育等途径。     

Kinetics of transfer of immunity by immune leucocytes and PFC response to HRBC in isogeneic ginbuna crucian carp

Transfer of immunity to horse erythrocytes (HRBC) by immune lymphoid cells was performed to analyze the kinetics of adoptive immunity in the clonal ginbuna crucian carp, Carassius auratus langsdorfti . Immune lymphoid cells were intravascularly transferred to the unprimed recipients and then recipients were evaluated by measuring the antibody titre of the plasma. Antibody productivity was most successfully conferred by splenic cells, followed by pronephric and mesonephric cells, taken from immune donors 7 days post-immunization, while transferability by thymic cells was lacking or very low, even if possible. Peak response of plaque-forming cells (PFCs) was observed at 5–7 days after the first injection, and the maximum number of PFCs at peak response was almost the same in all organs examined, such as the pronephros, mesonephros, spleen and the thymus. Direct correlation between transferability and number of PFCs was not observed on individuals, although the peak of transferability corresponded to that of the PFC response. Preliminary experiments of cell transfer by separated pronephric cells showed that the lymphocyte-rich fraction was more effective than the bottom fraction containing fewer lymphocytes in transferring immune reactivity. These results suggest that cells involved in transferring immune reactivity are B lymphocytes, composed of different developmental stages and distributed differently in the different lymphoid organs.     

草鱼头肾免疫细胞超微结构的观察

光镜和电镜观察表明,草鱼头肾器官含有大量的免疫细胞,主要包括淋巴细胞、浆细胞、粒细胞、单核细胞和巨噬细胞等。其中粒细胞根据细胞质内的颗粒的形态结构和大小,又分为Ⅰ型、Ⅱ型和Ⅲ型粒细胞。       

Peroxisomes in permanent and provisional kidneys

Peroxisomes in three forms of vertebrate kidney (pronephros, mesonephros, and metanephros), as permanent or provisional kidney, are summarized concerning their ultrastructure and developmental changes. Because the peroxisome is known to be diverse in mammalian metanephros, and species difference is its distinctive feature among cell organelles, information should be obtained on each kidney of each species. The ultrastructural and biochemical features of peroxisomes have at least been partly delineated in the metanephros and mesonephros, but nothing is known about the pronephros. Ultrastructural studies of the metanephric peroxisomes are present in mammals, birds, and reptiles, but information on their development is restricted to mammals and birds. As for the mesonephric peroxisomes, both ultrastructural and developmental data have been accumulating on mammals and amphibians, and ultrastructural information is present on fishes, but not on birds and reptiles. At present, studies on peroxisomes of provisional kidney have been restricted to mammalian mesonephros. The common features of renal peroxisomes previously examined are that they are spherical cell organelles with a single limiting membrane in ultrastructure, and are positive for catalase. Information on the ultrastructure and enzymes is not sufficient at present for comparing the ontogenesis of renal peroxisomes with their phylogenesis. Part of this study appeared as an original paper (11), and was presented as a poster at the International Symposium/CREST Research Conference, Peroxisome: Biogenesis, Function and Disease.     

二龄草鱼脾脏、肝脏组织高表达甘露糖结合凝集素mRNA

Innate immunity is expected to be very important in fish. Mannose-bingding lectin (MBL) participates in the innate immune system as an activator of the complement system and as an opsonin after binding to certain carbohydrate structures on microorganisms. In this experiment, total mRNA was isolated from spleen, liver, gills, thymus, head kidney and kidney of adult and immature grass carp Ctenopharygodon idllus. The cDNA of MBL was obtained by RT-PCR using total mRNA from the spleen of carp as template. Such cDNA was labled with ^32p and used as probe for Northern analysis, and autoradiographic signals were quantified by densitometry analysis. The results showed that MBL was high expressed in the spleen and liver and low in gills, thymus, head kidney and kidney of adult grass carp, and MBL was much lower expressed in spleen and liver of immature grass carp than those of adult grass carp. The results might partially explain why immature grass carp are vulnerable to grass carp hemorrhage virus (GCHV) whereas adult grass carp are not.This suggested that MBL mav be an imoortant anti-GCHV factor [Acta Zoologica Sinica 50 (1): 137 - 140. 2004].     

Kidney development conserved over species: essential roles of Sall1

The kidney develops in three stages: pronephros, mesonephros, and metanephros. Molecular mechanisms underlying these three steps are similar, and we can thus examine genetic cascades occurring during development. The induction system for pronephros in vitro has been established in Xenopus. Using this system, we isolated Sall1 that is essential for the initial step in metanephros formation. The potential mechanisms involved and future directions regarding kidney development are discussed.     

A high-efficiency,two-dimensional gel electrophoresis platform for mature leaves of grass pea (<Emphasis Type="Italic">Lathyrus sativus</Emphasis> L.)

Grass pea (Lathyrus sativus L.) is the most drought-tolerant legume crop rich in dietary protein. However, little is known about the molecular mechanisms of its drought resistance. Two-dimensional gel electrophoresis (2-DE) is an important experiment technique in proteomics, which has been applied extensively in studies on plant resistance to abiotic stress. To establish an effective 2-DE platform and further study the drought-resistance mechanisms of grass pea using proteomic approaches, three protein extraction methods, different isoelectric focusing (IEF) conditions and various types of gel strips were evaluated using mature leaves. The results showed that the trichloroacetic acid (TCA)/acetone protein extraction method, extending time at low voltage for IEF and using 18 cm gel strip with pH 4.0–7.0 were optimum conditions for 2-DE analysis of grass pea leaves. Applying these optimized 2-DE conditions, 1,481 total protein spots were detected in control leaves and 1,346 spots in polyethylene glycol -treated leaves, of which 67 differentially expressed protein spots were obtained relative to the control. These data suggested that an efficient 2-DE platform with high repeatability and resolution for grass pea mature leaves had been established for the first time here, which could be further used to investigate the drought-resistance molecular mechanisms of grass pea.     

Ontogeny of ENaC expression in the gills and the kidneys of the Japanese black salamander (Hynobius nigrescens Stejneger)

A full-length cDNA cloning and tissue distribution of epithelial sodium channel (ENaC) protein were studied during ontogeny by immunohistochemistry in the external gills, and the kidney, pronephros and mesonephros, of the Japanese black salamander, Hynobius nigrescens (Family Hynobiidae; a primitive caudate species). The amino acid sequence of Hynobius ENaCα is 64 and 63% identical to Bufo ENaCα and Rat ENaCα, respectively. In aquatic larva salamander at the digit differentiation stage, Hynobius ENaCα mRNA was expressed in the external gills and pronephros. In the adult, the mRNA was expressed in the skin and the mesonephros. In the larvae, juvenile, and adult specimens, Hynobius ENaCα immunoreactivity was observed at the apical cell membrane of the external gills, late parts of the distal tubules, and mesonephric duct in the kidney. Colocalization of the apical Hynobius ENaCα and the basolateral Na(+) ,K(+) -ATPase was observed in the tubular cells of pronephros and mesonephros. These results suggest that Hynobius ENaCα plays an important role in the regulation of sodium transport in the external gills and pronephros of aquatic larvae, and in the skin and mesonephros of terrestrial adult. This is the first study to indicate ENaC expression during ontogeny in amphibians. Since no orthologs or paralogs for ENaC have been found, so far, in databases of the genomes of teleosts, it is assumed that ENaC might have played a role in terrestriality during the evolution of early tetrapods, the origin of lissamphibians.     

病毒感染草鱼胸腺的EST分析和免疫相关基因的鉴定

以感染草鱼(Ctenopharyngodon idellus)出血病病毒(GCHV)的草鱼胸腺为材料,构建了草鱼胸腺的SMARTcDNA文库.筛选文库获得到1933条有效EST序列.BLASTX分析显示,583条序列在公共数据库中能找到同源基因(E-value≤1.00E 10-3,Identities≥30%),另外1350条序列则找不到显著同源性.已知基因按具体功能可划分为6类,大部分与细胞内的各种生理过程、细胞结构以及免疫防御相关.研究结果从分子水平上表明鱼类的胸腺在机体感染病毒的免疫反应中发挥重要作用,同时也表明胸腺组织在病毒感染后可能表达很多目前还不清楚功能的新基因.     

Changes in the protein spectrum of the chick embryo mesonephros and metanephros in the course of development

The protein spectra of two fractions (the soluble and the membrane fraction) of chick embryo kidney homogenates were isolated by electrophoresis on polyacrylamide gels with the aim of detecting the kidney differentiation process at the molecular level and, at the same time, of evaluating similarities in the construction of the mesonephros and metanephros at this level. Corresponding stages of the above two types of kidney were chosen for studying changes in protein structure during differentiation--i.e. the outset of differentiation (the 6-day mesonephros, the 11-day metanephros) and the stage of full maturity (the 14-day mesonephros, the 20-day metanephros). A total of 36 proteins was distinguished. The analysis of the protein spectra showed that the number of proteins changes but slightly during differentiation; the protein composition of the two types of kidney during differentiation altered by 20-35% of the total number of proteins; the similarity of the protein composition of the corresponding stages of mesonephros and metanephros, expressed as the proportion of the number of identical proteins, was greater than the mutual similarity of different developmental stages of the same type of kidney. The percentage of different proteins at corresponding stages of the kidneys varied from 5% to 23% of the total number of proteins detected.