应用iTRAQ标记技术结合2DLC-MS/MS分析家蝇幼虫免疫诱导后血淋巴蛋白质组

目的分析家蝇幼虫免疫诱导前后血淋巴中的免疫防御相关靶标,探索其先天性免疫机制。方法采用同位素标记及相对定量技术(isobaric tags for relative and absolute quantification,iTRAQ)结合2D LC-MS/MS对家蝇幼虫诱导前后的血淋巴蛋白质组进行研究。结果与对照组比较研究后共获得237个不同肽段,鉴定到13个具有定量信息的差异蛋白。免疫刺激后显著上调的蛋白有9个,下调蛋白4个(P0.05),对鉴定到的差异蛋白进行理化性质分析和GO(gene ontology)注释分析,发现这些蛋白分别具有抗菌、抗氧化、催化结合等功能,并参与了免疫、代谢、应激、转运等生物学过程,表明对家蝇幼虫经诱导后血淋巴中多种功能蛋白的表达发生了变化。结论 iTRAQ标记技术结合2D LC-MS/MS可以有效地分离鉴定昆虫血淋巴蛋白质组,为深入研究差异蛋白的功能奠定了基础。     

微管微丝交联因子1的结构与功能

微管微丝交联因子1(microtubule actin cross-linking factor 1,MACF1)是一种新的细胞骨架交联蛋白,属于血影斑蛋白(spectraplakin)家族成员之一,包含3个基本结构域即N末端结构域、杆状结构域及C末端结构域.其主要功能是交联微丝微管细胞骨架,参与细胞信号转导、蛋白质运输、胚胎发育以及疾病发生等过程.近年来,MACF1在细胞骨架动力学过程中的作用备受关注.现就该分子的结构与功能的最新研究进展进行综述.     

多功能的胞内衔接蛋白syntenin

Syntenin蛋白是在原核生物及真核生物中广泛存在的一类胞内衔接蛋白（adaptor proteins）. Syntenin由N端结构域（N-terminal domain,NTD）、两个串联的PDZ结构域(postsynaptic density protein, disc large and zonula occludens, PDZ)和C端结构域（C-terminal domain,CTD）组成,在生物进化过程中相对保守. Syntenin蛋白的PDZ结构域可与不同膜受体C端的PDZ结合基序（PDZ-binding motif,PBM）特异性结合, PDZ结构域结合受体的多样性导致了syntenin功能的多样性. 本文综述了syntenin蛋白的发现与分布及其结构特征,对syntenin在肿瘤转移、细胞质膜蛋白组装、参与动物免疫等领域的研究成果进行了较为详细的综述,同时介绍了syntenin在参与动物胚胎发育调控、血管生成和轴突生长等方面的研究进展.     

昆虫抗菌蛋白基因转录调控研究的新进展

在自然或人为创伤和感染情况下,昆虫能迅速产生各种类型的抗菌因子,例如天蚕素(cecropin),果蝇抗菌蛋白(diptericin),天蚕抗菌蛋白(attacin)和防御素(defensin)等.这些活性多肽和蛋白从其被合成的脂肪体和某些血细胞中,分泌到血淋巴参与虫体对入侵物的免疫反应.抗菌多肽和蛋白的诱导、表达及其协同作用于外源微生物,构成昆虫先天性防卫免疫系统中极为重要的环节.近年的研究表明,昆虫的这种防卫免疫系统与哺乳动物急性期反应是相关的,特别是在有关基因表达的协调控制方面具有许多共同的基本特征.     

融合蛋白CBD-SPA“Z”基因的构建、表达与活性鉴定

针对金黄色葡萄球菌蛋白A(SPA)层析柱的应用问题,将SPA的Z结构域基因与纤维素结合结构域(CBD)重组,并在Z结构域C端引入1个半胱氨酸(Cys),构建并表达出一种新型免疫亲和材料。利用基因工程技术将质粒pEZZ18中的Z基因插入到含有CBD的质粒pET35b(+)质粒中,构建出原核表达载体pET35b(+)-ZCys,经IPTG诱导表达,获得CBD-Protein Z融合表达蛋白。pET35b(+)-Z-Cys在E.coli BL21中正确表达,所表达的融合蛋白具有与哺乳动物IgG抗体结合的生物学活性和与纤维素结合的活性。结果证明CBD-Protein Z蛋白具有Z结构域与CBD结构域的活性,预计能在免疫亲和层析中用于IgG抗体的纯化。     

FⅧ的结构与血友病甲

凝血因子Ⅷ(FactorⅧ,F_Ⅷ)先天性缺陷而引起血友病甲,是临床上常见的一种遗传性出血性疾病.F_Ⅷ基因位于X染色体上,全长186kb,包括26个外显子及25个内含子.F_Ⅷ是一个分子量很大的糖蛋白(分子量为320kD),由具有相同N末端氨基酸顺序且分子量为90—200kD之间不同大小的一条重链和一条分子量为80kD的轻链组成,二链之间通过金属离子连结。在血浆中F_Ⅷ与血管性假血友病因子(von Willebrand factor     

菜粉蝶丝氨酸蛋白酶基因Pr-SP1的克隆及其表达谱分析

昆虫主要依靠先天免疫反应来抵御外源异物的入侵, 而与血淋巴黑化及抗菌肽合成等过程密切相关的丝氨酸蛋白酶激活级联反应在其中起着重要作用。为阐明丝氨酸蛋白酶在菜粉蝶Pieris rapae免疫中的作用, 本文通过简并引物RT-PCR克隆获得了菜粉蝶丝氨酸蛋白酶家族基因Pr-SP1的cDNA片段, 并利用RACE法扩增获得其全序列。该cDNA序列长1 489 bp, 其中开放阅读框长1 059 bp, 共编码353个氨基酸残基。Pr-SP1含一长度为20个氨基酸残基的信号肽序列, 其蛋白理论分子量为36.85 kDa, 理论等电点为6.41。多序列比对结果表明, Pr-SP1与其他昆虫的同源蛋白基因序列上存在较高一致性, 在N端有一个发夹结构域, 而C端是一个具有催化活性的结构域。实时荧光定量RT-PCR及免疫印迹结果表明, 蛹期Pr-SP1主要在颗粒血细胞内进行转录, 其蛋白产物主要定位在血浆; Pr-SP1在不同虫态及虫龄都有转录, 其蛋白产物在不同虫态及虫龄都有表达, 其中5龄幼虫最高, 卵期最低; Pr-SP1的转录水平及其蛋白产物的表达水平均会被大肠杆菌Escherichia coli、 藤黄微球菌Micrococcus luteus和巴斯德毕赤酵母Pichia pastoris诱导。根据这些结果分析认为, Pr-SP1属于Spätzle蛋白酶前体激活酶, 并参与菜粉蝶的先天免疫反应。     

耳聋相关基因COCH的非同义单核苷酸多态性致聋表型预测

群体凝血因子C同源物基因(Coagulation factor C homology,COCH)是人类发现的第一个伴前庭功能障碍的耳聋基因,位于人类染色体14q12-q13上。迄今,在COCH基因上发现16个位点突变导致常染色体显性遗传非综合征型耳聋DFNA9的发生,其中包括13个非同义单核苷酸多态性(Non-synonymous single nucleotide polymorphisms,nsSNPs)位点。由于该基因其他nsSNPs的基因型与表型关系尚不清楚,因此文章采用生物信息学方法,从COCH基因全部的SNPs中分级筛选,结合已知的致病nsSNPs信息及蛋白三维结构验证,首次预测出由COCH基因编码的cochlin蛋白的vWFA (Von Willebrand factor type A domain)区的8个高风险致病性nsSNPs(I176T、R180Q、G265E、V269L、I368N、I372T、R416C和Y424D)。同时,对位于LCCL (Limulus factor C, cochlin, and late gestation lung protein Lgl1)区域的6个已知致病突变的nsSNPs ( P51S、G87W、I109N、I109T、W117R和F121S)进行了三维结构模拟,发现突变体均发生了环状结构或链状结构的改变。本研究对COCH基因的基因型与表型的相关性研究为遗传性耳聋筛查提供了相应的理论依据,也对该基因所编码的cochlin蛋白的功能研究具有一定的指导意义。     

家蚕C类清道夫受体BmSR-C的基因克隆、原核表达及亚细胞定位

【目的】本研究旨在克隆与鉴定家蚕Bombyx mori C类清道夫受体基因BmSR-C,为探析其在家蚕免疫中的功能奠定基础。【方法】利用RACE技术克隆家蚕C类清道夫受体基因全长序列,并对其进行生物信息学分析。利用RT-PCR和qPCR方法对BmSR-C基因的时空表达情况进行检测。通过原核表达和Ni~+亲和层析的方法获得BmSR-C重组蛋白,免疫昆明小鼠制备抗BmSR-C多克隆抗体。利用ELISA法和Western blot分别对鼠抗BmSR-C蛋白多克隆抗体的效价和特异性进行检测。构建家蚕BmSR-C的真核表达载体,转染家蚕BmE细胞,分析该蛋白的亚细胞定位情况。【结果】克隆获得家蚕BmSR-C基因(GenBank登录号:BGIBMGA004577),其开放阅读框(ORF)全长为1 821 bp,编码606个氨基酸残基。BmSR-C具有典型的C类清道夫受体家族结构特征,主要由CCP, MAM和SO结构域以及靠近C端的单次跨膜结构域组成。进化分析结果显示鳞翅目昆虫SR-C单独聚为一支,家蚕BmSR-C与同为鳞翅目昆虫的草地贪夜蛾Spodoptera frugiperda和大红斑蝶Danaus plexippus的同源蛋白亲缘关系最为接近。对BmSR-C基因的时空表达分析表明,BmSR-C在家蚕的马氏管和血细胞中高表达,而在其他组织中无明显表达;其在家蚕不同发育时期的血细胞中均有表达,且在4龄眠期的表达量达到峰值。ELISA检测结果显示,所制得抗体效价高达1∶128 000;Western blot检测结果显示,该抗体可以特异性识别重组蛋白。家蚕BmE细胞中的亚细胞定位结果表明BmSR-C主要定位于细胞膜。【结论】获得家蚕C类清道夫受体基因BmSR-C的完整cDNA序列及其表达特征;成功制备了BmSR-C的多克隆抗体,利用家蚕BmE细胞在细胞水平上分析了BmSR-C的亚细胞定位情况,推测其参与家蚕的生长发育及病原微生物入侵的免疫反应,为进一步研究BmSR-C的生物学功能奠定了基础。     

昆虫血蓝蛋白功能研究进展

血蓝蛋白是一种重要的昆虫呼吸蛋白,参与昆虫的氧气转运、免疫防御和蛋白存储等多种生理过程,并显著影响昆虫的生长发育及其对环境的适应性.近年来,血蓝蛋白在不完全变态昆虫中被陆续报道;血蓝蛋白的进化及功能已受到国内外学者的广泛关注.基于目前研究现状,本文系统综述昆虫血蓝蛋白的结构和生物学功能,并重点探讨血蓝蛋白对昆虫氧气转运和低氧适应的影响.     

Host-derived extracellular nucleic acids enhance innate immune responses, induce coagulation, and prolong survival upon infection in insects

Extracellular nucleic acids play important roles in human immunity and hemostasis by inducing IFN production, entrapping pathogens in neutrophil extracellular traps, and providing procoagulant cofactor templates for induced contact activation during mammalian blood clotting. In this study, we investigated the functions of extracellular RNA and DNA in innate immunity and hemolymph coagulation in insects using the greater wax moth Galleria mellonella a reliable model host for many insect and human pathogens. We determined that coinjection of purified Galleria-derived nucleic acids with heat-killed bacteria synergistically increases systemic expression of antimicrobial peptides and leads to the depletion of immune-competent hemocytes indicating cellular immune stimulation. These activities were abolished when nucleic acids had been degraded by nucleic acid hydrolyzing enzymes prior to injection. Furthermore, we found that nucleic acids induce insect hemolymph coagulation in a similar way as LPS. Proteomic analyses revealed specific RNA-binding proteins in the hemolymph, including apolipoproteins, as potential mediators of the immune response and hemolymph clotting. Microscopic ex vivo analyses of Galleria hemolymph clotting reactions revealed that oenocytoids (5-10% of total hemocytes) represent a source of endogenously derived extracellular nucleic acids. Finally, using the entomopathogenic bacterium Photorhabdus luminescens as an infective agent and Galleria caterpillars as hosts, we demonstrated that injection of purified nucleic acids along with P. luminescens significantly prolongs survival of infected larvae. Our results lend some credit to our hypothesis that host-derived nucleic acids have independently been co-opted in innate immunity of both mammals and insects, but exert comparable roles in entrapping pathogens and enhancing innate immune responses.     

Hemostasis in larvae of Manduca sexta: formation of a fibrous coagulum by hemolymph proteins

Little is known of the participation of insect hemolymph proteins in wound healing and clot formation. We describe the assembly of purified hemolymph protein from the tobacco hornworm into an extended fibrous coagulum in the absence of hemocytes. This coagulum resembles the clot formed from bovine fibrinogen and thrombin. Structural components of the coagulum are present in hemolymph, however, spontaneous assembly occurs only in hemolymph collected through a wound. The fibrous coagulum assembles from purified structural protein(s) following addition of a non-protein factor from hemolymph, which is also present in Grace's insect cell culture medium.     

Proteomic analysis of the Drosophila larval hemolymph clot

Components of the insect clot, an extremely rapid forming and critical part of insect immunity, are just beginning to be identified (1). Here we present a proteomic comparison of larval hemolymph before and after clotting to learn more about this process. This approach was supplemented by the identification of substrates for the enzyme transglutaminase, which plays a role in both vertebrate blood clotting (as factor XIIIa) and hemolymph coagulation in arthropods. Hemolymph proteins present in lower amounts after clotting include CG8502 (a protein with a mucin-type domain and a domain with similarity to cuticular components), CG11313 (a protein with similarity to prophenoloxidase-activating proteases), and two phenoloxidases, lipophorin, a secreted gelsolin, and CG15825, which had previously been isolated from clots (2). Proteins whose levels increase after clotting include a ferritin-subunit and two members of the immunoglobulin family with a high similarity to the small immunoglobulin-like molecules involved in mammalian innate immunity. Our results correlate with findings from another study of coagulation (2) that involved a different experimental approach. Proteomics allows the isolation of novel candidate clotting factors, leading to a more complete picture of clotting. In addition, our two-dimensional protein map of cell-free Drosophila hemolymph includes many additional proteins that were not found in studies performed on whole hemolymph.     

Helix pomatia lectin and annexin V,two molecular probes for insect microparticles: possible involvement in hemolymph coagulation

Insect hemolymph coagulation involves a complex reaction with contributions from hemocytes and soluble factors. Here we present evidence for the presence of microparticles in the coagulation reaction. These particles are formed by hemocytes in a calcium-dependent process. Both the particles and the remaining cells are labelled by annexin V indicating the presence of phosphatidylserine on the outer membrane. Microparticles are enriched in hemomucin, a surface protein of Drosophila hemocytes that is specifically recognised by a snail (Helix pomatia) lectin. Hemomucin is shown to bind to lipophorin, a multifunctional hemolymph molecule previously implied in coagulation. Our findings suggest similarities at a biochemical and cellular level between vertebrate blood and insect hemolymph coagulation.     

Eicosanoid actions in insect cellular immune functions

Insects are more or less constantly challenged with a daunting array of pathogenic organisms, including viruses, bacteria, fungi, protozoans as well as various metazoan parasites and parasitoids. At the first level of defense, the pathogens are rebuffed by physical barriers, including the cuticle and peritrophic membrane. Upon breaching these barriers, pathogens meet with an arsenal of robust and efficacious immune defense mechanisms. Two general categories of defenses are typically recognized, humoral defenses and hemocytic or cellular defenses. The former involves induced synthesis of various antibacterial proteins and peptides, such as cecropins and lysozyme. Cellular defense mechanisms are characterized by direct interactions between circulating hemocytes and the invaders. These include phagocytosis, microaggregation, nodulation, and encapsulation. Microaggregation is a step in the nodulation process, which is responsible for clearing the bulk of bacterial infections from circulation. Coordinated cellular actions lead to encapsulation of invaders, such as parasitoid eggs, that are very much larger than individual hemocytes. While the defense mechanisms are broadly appreciated, less is known about the biochemical signals responsible for mediating and coordinating the cellular actions. We now know eicosanoids mediate phagocytosis, microaggregation, and nodulation reactions to immune challenge, as well as cell spreading, a specific step in nodulation. We have several goals in this mini review. We provide a brief background on cellular immunity, outline eicosanoid biosynthesis, and review eicosanoid actions in cellular immunity in insects. Recent work indicates some pathogens have usurped eicosanoid‐mediated immunity; they disable insect immunity by inhibiting eicosanoid biosynthesis. We interpret these findings and their significance with respect to the biological control of insects. We also present preliminary work designed to test hypotheses on how eicosanoids exert their actions. We address shortcomings in our knowledge on eicosanoids in insect biology.     

The relative abundance of hemocyte types in a polyphagous moth larva depends on diet

Hemocytes are crucial cells of the insect immune system because of their involvement in multiple immune responses including coagulation, phagocytosis and encapsulation. There are various types of hemocytes, each having a particular role in immunity, such that variation in their relative abundance affects the outcome of the immune response. This study aims to characterize these various types of hemocytes in larvae of the grapevine pest insect Eupoecilia ambiguella, and to assess variation in their concentration as a function of larval diet and immune challenge. Four types of hemocytes were found in the hemolymph of 5th instar larvae: granulocytes, oenocytoids, plasmatocytes and spherulocytes. We found that the total concentration of hemocytes and the concentration of each hemocyte type varied among diets and in response to the immune challenge. Irrespective of the diet, the concentration of granulocytes increased following a bacterial immune challenge, while the concentration of plasmatocytes and spherulocytes differentially varied between larval diets. The concentration of oenocytoids did not vary among diets before the immune challenge but varied between larval diets in response to the challenge. These results suggest that the resistance of insect larvae to different natural enemies critically depends on the effect of larval diet on the larvae’s investment into the different types of hemocytes.     

昆虫天然免疫相关基因研究进展

在长期进化过程中,昆虫形成了强大的天然免疫防御系统,即体液免疫和细胞免疫。体液免疫主要包括Toll、IMD和JAK/STAT 3条信号通路,通过信号转导及免疫途径调控免疫相关基因的表达,诱导产生抗菌肽和其他效应分子。细胞免疫由血细胞介导,主要完成对病原物的包裹、吞噬和集结等。近年来,昆虫基因组学快速发展,通过生物信息学等方法从昆虫基因组数据中已鉴定到大量免疫相关基因,对这些基因的研究加深了人们对昆虫天然免疫分子机制的认识和理解。根据基因功能,免疫相关基因分为识别、信号转导、调制器、效应分子、黑化反应、RNA干扰和其他基因等7类,这些基因通过互作来调控体液免疫和细胞免疫。本文对昆虫免疫相关基因的分类、功能及家族进化等方面的研究成果进行总结,并对今后昆虫免疫的研究重点进行了展望,以期为昆虫免疫分子机制的研究及开发新的害虫防治策略提供依据。     

昆虫造血作用和造血干细胞研究进展

昆虫血细胞（insect hemocyte）在昆虫代谢、 发育变态以及先天免疫等方面承担着重要的作用。昆虫只有先天免疫系统, 血细胞所行使的免疫功能对于昆虫对抗外源病菌尤为重要。本文主要介绍了昆虫血细胞类型、 造血作用、 造血干细胞及造血相关因子的相关研究。通过特殊染色和形态学观察, 果蝇Drosophila血细胞主要由3类细胞组成, 而鳞翅目等大部分昆虫血细胞由5类细胞组成。昆虫血细胞主要存在于循环血液环境及造血器官内, 而在这两个系统中都存在有进行复制的血细胞, 这为研究昆虫造血干细胞特性和其定位提供了一个很好的系统。果蝇血细胞祖细胞来自于胚胎中胚层细胞, 然后再分化为各种血细胞, 这一系列分化过程由造血因子所调控。     

Insect hemolymph clotting: evidence for interaction between the coagulation system and the prophenoloxidase activating cascade

Here we describe a novel approach to isolate proteins involved in insect hemolymph coagulation. In order to avoid problems in purifying clot proteins after they had been crosslinked, we performed an in vitro coagulation reaction with cell-free hemolymph from the lepidopteran Galleria mellonella and used the resulting complexes to produce a specific antiserum. The antiserum reacted with a subset of hemolymph proteins as well as with granular cells, but not with other hemocyte types of Galleria. Screening expression libraries identified some positive clones, which turned out to code for some previously characterized components of immune cascades, as well as some novel candidates for clotting factors. Known components include members of both the coagulation system and the prophenol-activating cascade, lending support to the idea that both systems work together during the formation of a hemolymph clot. Novel candidates for insect clotting factors include a mucin-like protein, a glutathione-S-transferase, and a distant member of the alpha-crystallin/small heat shock protein family. Using assays measuring the activity of transglutaminase, a key enzyme in clotting reactions in both vertebrates and invertebrates, we found a partial overlap between transglutaminase substrates and proteins recognized by the antiserum against the in vitro-induced clot.