不同宿主来源日本血吸虫童虫差异表达基因的研究
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摘要
血吸虫病是一种分布广泛、危害严重的人畜共患寄生虫病。已报道在我国流行的日本血吸虫中国大陆株可感染40余种哺乳动物,日本血吸虫对不同宿主有着不同的感受性,如在小鼠等易感宿主体内血吸虫的发育率达60-70%,在大鼠等非易感宿主体内的发育率为10-20%,而血吸虫感染东方田鼠后,大部分虫体在感染后2周死亡,不能发育成熟。东方田鼠是至今发现的唯一一种感染血吸虫后不致病的哺乳动物,查明其抗血吸虫病机理具有重要的理论和实际意义。此前该领域相关研究多限于形态观察、感染、致病和免疫现象等,研究亟待深入发展。为此,本研究以实验室感染的来源于易感宿主BALB/c小鼠,非易感宿主Wistar大鼠及不致病宿主东方田鼠的日本血吸虫10d童虫为研究对象,应用日本血吸虫寡核苷酸芯片技术比较、分析三种不同宿主来源的日本血吸虫基因表达方面的差异。并对东方田鼠来源虫体呈低表达的凋亡抑制基因Sj IAP和呈高表达的凋亡相关基因caspase3和caspase 7;在小鼠来源虫体呈高表达的生长发育、信号传导相关基因Sj MAP 2、Sj mago nashi、Sj Cyclophilin A、Cyclophilin B和Cyclophilin C等进行克隆、表达,及生物学功能初步分析,旨在揭示在不同宿主环境中,日本血吸虫生长发育差异的分子基础。研究结果对阐明血吸虫生长发育机制、与宿主相互作用机理,发现血吸虫生长发育相关的重要分子,也可为研制血吸虫病疫苗、新治疗药物和对血吸虫病防治提供新思路和新途径。
1、不同宿主来源日本血吸虫10d童虫形态观察及体细胞凋亡分析
实验室中以日本血吸虫尾蚴感染小鼠、大鼠和东方田鼠,10d后收集虫体。光镜下观察到不同来源日本血吸虫形态大小有很大差异,东方田鼠来源童虫长402.2±102.2μm,宽159.1±47.3μm,大鼠来源童虫长828.3±127.4μm,宽103.4±22.8μm,小鼠来源童虫长878.5±137.4μm,宽88.3±20.0μm。扫描电镜下观察,东方田鼠来源童虫虫体萎缩,口吸盘形成空泡,表面结构消失,腹吸盘表面结构改变,体表正常棘消失,萎缩,形成空泡;小鼠和大鼠来源虫体体表棘等结构无异常变化。透射电镜观察发现,东方田鼠来源童虫虫体细胞结构受到破坏,细胞崩解,细胞核仁消失,染色质凝集,有细胞凋亡的表现。小鼠和大鼠来源虫体细胞形态正常,细胞核和线粒体无明显变化。利用Annexin V-FITC/propidium iodide (PI)分析不同宿主来源虫体细胞凋亡状况表明,小鼠来源虫体的凋亡细胞比例为0.40%;大鼠来源虫体的凋亡细胞比例为5.22%;东方田鼠来源虫体的凋亡细胞比例为62.91%,表明东方田鼠来源虫体的细胞发生了明显凋亡。本研究首次利用扫描电镜和透射电镜技术,进行东方田鼠、大鼠和小鼠来源日本血吸虫童虫显微和超微结构的观察和比较,利用流式细胞仪检测发现日本血吸虫在不同宿主体内的生长发育状况与体细胞凋亡相关。
2、不同宿主来源日本血吸虫童虫基因表达的芯片分析
应用寡核苷酸芯片技术,比较分析小鼠、大鼠和东方田鼠来源的日本血吸虫10d童虫的基因表达差异。结果表明,与小鼠来源童虫相比,东方田鼠来源童虫有3293个基因呈下调表达(fold<0.5),71个基因呈上调表达(fold>2);大鼠来源童虫有3335个基因呈下调表达(fold<0.5),133个基因呈上调表达(fold>2)。其中,东方田鼠来源童虫显著性下调表达基因81个(fold<0.2),显著性上调表达基因18个(fold>5);大鼠来源童虫显著性下调表达基因210个(fold<0.2),显著性上调表达基因54个(fold>5)。东方田鼠和大鼠来源童虫具有相似表达趋势的显著性下调表达基因有27个(fold<0.2),显著性上调表达基因有7个(fold>5)。
对显著性差异表达基因进行GO分类和KEGG通路等生物信息分析,结果显示,东方田鼠来源童虫显著性上调表达基因主要与细胞(cell)、细胞凋亡(apoptosis)、细胞外组成部分(extracellular region part)、细胞组成(cell part)、酶调节活性(enzyme regulator activity)和转录调节活性(translation regulator activity)等相关。东方田鼠来源童虫显著性下调表达基因主要与代谢过程(metabolic process)、催化活性(catalytic activity)、发育过程(developmental process)、转运活性(transporter activity)、细胞进程(cellular process)、定位(localization)和结合(binding)等相关。东方田鼠和大鼠来源童虫均显著下调表达基因与代谢过程(metabolic process)、定位(localization)、结构形成自动修饰(anatomical structure formation)和催化活性(catalytic activity)等相关。对东方田鼠和大鼠来源童虫均显著下调表达的基因进行KEGG pathway通路分析,发现这些蛋白主要为甘氨酸/苏氨酸/丝氨酸代谢分子(Glycine,serine and threonine metabolism)、DNA复制关键分子(DNA replication)、MAPK信号通路分子(MAPK signaling pathway)、蛋白转运(Protein export)、氨酰基tRNA生物合成(Aminoacyl-tRNA biosythesis)等分子。本研究发现一批基因在三种宿主来源10d童虫中呈现差异表达,它们可能是不同宿主来源童虫发育差异的关键分子,影响血吸虫在不同宿主中的发育。如东方田鼠来源童虫显著上调表达,小鼠来源虫体显著下调的基因有与细胞增殖密切相关的颗粒蛋白(Granulin)基因,细胞凋亡通路中发挥凋亡效应的关键分子如Cell death protein 3,Caspase 7,caspase 3;在小鼠来源童虫显著上调表达,东方田鼠来源童虫显著下调表达的基因中与生长发育相关的甲硫氨酰氨肽酶2 (MAP 2)和Sj magonashi基因,与胰岛素代谢相关的胰岛素分子(Insulin-2)和胰岛素受体蛋白激酶(insulin receptor protein kinase)基因,与脂肪酸代谢相关的脂肪酸去饱和酶(Fatty acid desaturase)、长链脂肪酸延伸相关蛋白(Elongation of very long chain fatty acids protein)、脂肪酸脱氢酶(Fatty-acid amide hydrolase)、脂肪酸结合蛋白(fatty acid-binding protein)基因,与细胞凋亡通路中发挥抑制凋亡效应的关键分子相关Sj IAP(Baculoviral IAP repeat-containing protein)、cytokine-induced apoptosis inhibitor,与信号传导相关的Cyclophilin家族基因、TGF通路分子如Eukaryotic translation initiation factor 3,Transforming growth factor beta-1、Wnt通路分子Wnt inhibitory factor 1等。这些重要功能分子的发现,将为阐明血吸虫在不同宿主生存环境中的生长发育机制,探讨血吸虫与宿主相互作用关系、发现新的血吸虫候选疫苗分子和新药物靶标提供重要基础。
本研究分析获得了一批小鼠、大鼠和东方田鼠来源的10d童虫的差异表达基因信息,初步揭示了在不同宿主体内日本血吸虫生长发育的分子基础,发现了一批在不同宿主环境中可能影响血吸虫生长发育的关键功能分子,为这些重要差异表达基因的确定及进行生物学功能研究提供了良好的基础。
3、日本血吸虫细胞凋亡因子和凋亡抑制因子(Sj IAP)的研究
基因组水平的研究表明血吸虫与其他多细胞生物一样具有凋亡这一生物学现象。上文透射电镜研究表明,东方田鼠来源10d童虫细胞有细胞凋亡的现象,同时芯片分析结果表明,日本血吸虫凋亡相关基因caspase3和7在东方田鼠来源虫体中呈高表达,而在小鼠来源虫体中呈较低表达;日本血吸虫凋亡抑制因子基因在小鼠来源虫体中高表达,东方田鼠来源虫体中低表达,表明日本血吸虫凋亡相关基因和凋亡抑制因子可能在血吸虫生长发育和寄生虫与宿主相互作用关系中发挥重要的生物学功能,在东方田鼠抗血吸虫机制中发挥一定的作用。
本研究进行了不同宿主来源日本血吸虫童虫caspase3/7的活性和基因表达差异研究;利用RT-PCR技术获得了Sj IAP编码基因的全长cDNA,PCR扩增其mRNA对应基因组DNA序列;荧光实时定量PCR和western blot分析表明,该基因为成虫期雄虫期高表达基因;免疫组化研究分析表明,虫体IAP蛋白广泛分布于成虫体被膜;细胞水平实验证实该基因真核重组表达质粒在细胞水平可以有效抑制诱导剂引起的细胞凋亡,同时研究发现Sj IAP重组蛋白也可以在血吸虫的虫体水平抑制凋亡;Real Time RT-PCR和western blot研究了日本血吸虫凋亡抑制因子基因(IAP)在不同宿主的差异表达情况。
研究结果表明细胞凋亡相关基因很可能是不同宿主环境中影响血吸虫生长发育的重要功能分子,在血吸虫生长发育调控和东方田鼠抗血吸虫病中可能发挥重要的生物学功能。
4、日本血吸虫生长发育重要功能分子甲硫氨酰氨肽酶2基因(MAP 2)和mago nashi基因的研究
MAP 2是一种双功能酶,其催化结构域位于C端,负责切除蛋白质合成过程中新生肽链N端的起始蛋氨酸,对蛋白质的成熟以及在细胞中的转运和定位、功能调节极其重要,其N端结构域含有酸性氨基酸和碱性氨基酸富集的区域,可以抑制真核起始因子eIF2-2α的磷酸化,促进蛋白质合成的起始。在哺乳动物细胞内,MAP 2与细胞增值与分化密切相关,MAP 2在秀丽线虫生殖和发育中发挥重要的生物学功能。mago nashi基因在真核生物中广泛存在,且极为保守。在生物体翻译转录中参与mRNA定位和剪切,在生物体生殖与发育中,mago nashi基因在胚胎早期发育和生殖细胞性别决定中发挥重要的生物学功能。芯片分析结果表明,日本血吸虫MAP 2和mago nashi基因在东方田鼠来源虫体中低表达,大鼠和小鼠来源虫体中高表达。
本研究利用RT-PCR技术分别获得了Sj MAP 2(日本血吸虫甲硫氨酰氨肽酶2)和Sj mago nashi编码基因的全长cDNA(基因登录号为:GQ403663和GQ403668)。荧光实时定量PCR和western blot分析Sj MAP 2和Sj mago nashi基因在不同期别阶段虫体表达差异,发现这两个基因均为童虫期表达基因,且在雄虫的表达量均高于其在雌虫。Real Time RT-PCR和western blot分析Sj MAP 2和Sj mago nashi在不同宿主来源日本血吸虫童虫的表达情况,发现二者均在小鼠来源童虫中表达最高,大鼠次之,东方田鼠最低。应用吡喹酮处理雌虫和雄虫后,Sj M AP 2表达均有显著降低,其中雄虫和雌虫中分别下降92.17%和49.01%。构建了这2个基因的重组表达质粒,并分别在大肠杆菌中成功表达。Western blotting分析表明重组蛋白rSj MAP 2和rSj mago nashi具有良好的抗原性,应用这两种重组抗原免疫小鼠,与空白对照组比较,免疫组小鼠获得的减虫率分别为17.8%和22.43%,肝脏虫卵减少率分别为36.24%和29.11%。
以上研究表明,日本血吸虫Sj MAP 2和Sj mago nashi基因的表达可能对血吸虫生长发育有影响,这两个基因可能在血吸虫生殖、生长发育及细胞分化中具有重要的生物学功能。
5、日本血吸虫Cyclophilin家族CyPA、CyPB和CyPC基因的研究
Cyclophilin(CyP)广泛存在于生物体内,为一种分布广泛的细胞内蛋白,在植物、细菌和哺乳动物中均存在,具有高度保守性。CyP具有肽酰脯氨酰顺反异构酶(PPIase)活性,在体内外能结合蛋白质并催化加速它们的折叠、装配和转运;特别是富含脯氨酸的蛋白质折叠,起着分子伴侣的作用,同时在信号转导中起着重要调节作用。信号转导与蛋白翻译后修饰在日本血吸虫中发挥重要的生物学功能。
本研究利用RT-PCR技术分别获得了Sj CyP A、Sj CyPB和Sj CyPC编码基因的全长cDNA(基因登录号为:GQ403666、GQ403664和GQ403665)。荧光实时定量PCR分析这三个基因在不同期别阶段虫体表达量,发现这三个基因均为童虫期高表达基因。Real Time RT-PCR分别分析Sj CyP A、Sj CyPB和Sj CyPC在不同宿主来源日本血吸虫童虫的差异表达情况,发现三者均在小鼠来源童虫中表达最高,大鼠次之,东方田鼠最低。构建了这3个基因的重组表达质粒,并分别在大肠杆菌中成功表达Western blotting验证表明重组蛋白rSj CyP A、rSj CyPB和rSj CyP C均具有良好的抗原性,在小鼠免疫试验中,与空白对照组比较,三种蛋白免疫小鼠获得的减虫率分别为14.2%、26.98%和13.79%;肝脏虫卵减少率分别为43.38%、39.73%和51.32%。
研究表明,日本血吸虫Sj CyP A、Sj CyPB和Sj CyPC基因的表达可能对血吸虫生长发育有影响,这三个基因可能在血吸虫的分子伴侣、信号转导等方面发挥重要的生物学功能。
本文以日本血吸虫易感宿主BALB/c小鼠、非易感宿主Wistar大鼠和不致病宿主东方田鼠来源的10d童虫作为研究对象,应用电镜观察表明三种不同宿主来源的10d童虫在形态结构上存在较大差异。首次发现东方田鼠来源童虫虫体细胞的凋亡现象显著大于大鼠和小鼠来源虫体。首次应用寡核苷酸芯片技术对三种宿主来源童虫的差异表达基因进行了比较分析,发现一些重要的血吸虫生长发育、细胞凋亡、信号传导分子在三种不同宿主来源虫体呈差异表达,获得一批具有深入研究价值的差异表达基因信息。首次克隆、鉴定和表达了Sj MAP2, Sj magonashi, Sj CyPA, Sj CyPB和Sj CyPC五个血吸虫新基因,登录号分别为GQ403663、GQ403668 GQ403666、GQ403664和GQ403665。应用以上五种基因重组抗原进行了小鼠免疫试验,这5个基因的重组蛋白都诱导了部分免疫保护作用。首次对日本血吸虫凋亡抑制基因Sj IAP的生物学功能进行了探索,在细胞水平验证了该凋亡抑制因子可有效地抑制凋亡诱导剂诱发的凋亡。本文结果对深入探讨日本血吸虫的生长发育机制,发现血吸虫与宿主相互作用关键分子,进而为发现抗血吸虫病新的疫苗候选分子和新的治疗药物靶标提供了新思路。
Schistosomiasis is one of the wide spread and prevalent parasitic diseases worldwide. Nearly 40 kinds of animals can be infected by Schistosoma japonicum (Chinese mainland strain). The previous research had demonstrated that BALB/c mouse as a susceptible host of schistosome, Wistar rat as a unsusceptible host and Microtus fortis as resistant host of Schistosoma japonicum infection. Microtus fortis is the resistant host of Schistosoma japonicum. Animals have different symptoms after infected by cercaria: 60-70% of the adult Schistosoma japonicum can be perfused post-cercarial challenge from the host in BALB/c mouse, but 10-20% from Wistar Rat. Most of the schistosolums have incomplete development and fail to accomplish their life cycles in Microtus fortis. Till now, Microtus fortis is the only unmorbific host after infected with schistosoma japonicum. The microarray analysis of the gene expression difference between mouse, Wistar rat and Microtus fortis demonstrated that the genes related with immune mechanism, apoptosis, growth and development were differential expressed. Our research work focused on the different expression genes in 10 days Schistosolum from the hosts of Microtus fortis, BALB/c mouse and Wistar rat with the analysis of oligonucleotide microarray and bioinformatics techniques. A great number of genes related with signal transduction, immune escape, development and metabolism had been screen out with the analysis. Several genes of Schistosoma japonicum were selected for further research: genes referred with apoptosis such as Sj IAP、caspase3 and caspase 7, genes related with development and growth such as Sj MAP 2 and Sj mago nashi, in signal transduction such as Cyclophilin A,B and C genes.Our data presented here suggest that such genes may play important role in the development; reproduction and host-parasite interplay in schistosome living, and may be a potential new drugs or vaccine target for controlling schistosomiasis.
1、Scanning electron microscope(SEM) and transmission electron microscopy (TEM) observation of Schistosolum from the three different hosts
There are obvious changes in the development and growth of Schistosoma japonicum in different hosts. After two weeks post infected with cercaria, Schistosolum gradually withered and stopped growth, then died. The width and length of schistosomula were measured with optical microscope.The length of schistosomula from Microtus fortis, Wistar rat and BALB/c mouse was 402.2±102.2, 828.3±127.4 and 878.5±137.4. The width of schistosomula from Microtus fortis, Wistar rat and BALB/c mouse was 159.1±47.3,103.4±22.8 and 88.3±20.0.The morphologic comparion of the schistosolums from three different hosts with SEM and TEM showed that microscopic and ultrastructural structure of the schistosolums have significant difference. The surfaces of the schistosomula from Microtus fortis were stunted, shrinking and vacuole.The nucleus of the cells in schistosomula from Microtus fortis were agglutinated. All these revealed that the cells of Schistosolum may have the phenomenon of apoptosis. Schistosolum were divided into monoplast with trypsinization, and then apoptosis were determined by flow cytometry (FCM) with the analysis of Annexin V-FITC/propidium iodide (PI).
2、Analyze differentially expressed genes of 10 days schistosolum from the three different hosts
Based on the data of oligonucleotide microarray and bioinformatics analysis of the differentially expressed genes from the three different hosts: in the schistosolums from Microtus fortis,3293 genes were down-regulated (fold <0.5)and 71 genes were up-regulated (fold >2) in comparison with the schistosolums in BALB/c mouse; in the schistosolums in from Wistar rat , 3335 geneswere down-regulated and 133 genes were up-regulated in comparison with the schistosolums in BALB/c mouse.
Preliminary function analysis with Gene Ontology GO classification and KEGG pathways analysis indicate that the up-regulated expressed genes in the schistosolums from Microtus fortis focused on the function as cell, extracellular region part, cell part, enzyme regulator activity, translation regulator activity, the down-regulated expressed genes in the schistosolums from Microtus fortis focused on the function as metabolic process, catalytic activity, developmental process, transporter activity, cellular process, localization and binding. The significant down-regulated expressed genes (fold <0.2) in the schistosolums from both Microtus fortis and Wistar rat included metabolic process, localization, anatomical structure formation and catalytic activity. Also another some genes not related with the above process are also co-down regulated, such as serine/threonine-protein kinase, NAD(P)H-quinone oxidoreductase, Pericentriolar material, Cell surface receptor daf-4. The KEGG pathway analysis revealed that the co-down regulated expressed genes in the schistosolums from both Microtus fortis and Wistar rat consist of the pathway of Glycine, serine and threonine metabolism, DNA replication, MAPK signaling pathway, Protein export, Aminoacyl-tRNA biosythesis. Because the genomic research works of S. japonicum and S. mansoni were fall behind the Model organism Caenorhabditis elegans, we do the KEGG pathway analysis of the co-down regulated expressed genes with the C. elegans pathway model. We found that some genes are involved in the pathway as Apoptosis, Oxidative phosphorylation, Insulin signaling pathway and Fatty acid metabolism and so on.
The 10 days schistosolum specific expression genes were selected and analyzed with the gene expression of NADH dehydrogenase [ubiquinone] flavoprotein 2 as the internal control. The result showed that 1178 genes were down-regulated (fold <0.5) and 524 genes were up-regulated (fold >2), also 124 significant down-regulated genes (fold <0.2) and 578 significant up-regulated genes (fold >5). The result of the Gene Ontology GO classification analysis of the significant up-regulated/high genes demonstrated that the different function genes between these two trend genes focused on metabolic process, catalytic activity, enzyme regulator activity, transcription regulator activity, transporter activity, antioxidant activity, electron carrier activity, structural molecule activity. In the other part, the KEGG pathways of the different function genes are involved in Protein export, MAPK signaling pathway, Oxidative phosphorylation, Inositol phosphate metabolism, Calcium signaling pathway, Progesterone-mediated oocyte maturation), Ubiquitin mediated proteolysis, Notch signaling pathway and estrogen-related receptor beta like 2.
3、Analyzed of apoptosis in Schistosoma japonicum from different hosts and molecular characterizations of an inhibitor of apoptosis
Apoptosis is a normal process for regulating cellular death of many organisms. We molecularly characterized an inhibitor of apoptosis from Schistosoma japonicum (SjIAP). The transcription of the SjIAP predominantly occurred at the developmental stages in a final host. The immunohistochemistry analysis revealed that Sj IAP protein was located in the tegument of the adult male worm of schistosoma japonicum. Functional assay indicated that the SjIAP could inhibit caspase activity either in 293T cell or in schistosome lysates. Additionally, there were differently expressed profiles of the SjIAP in S. japonicum living in different hosts. The gene expression of two important molecules caspase 3 caspase 7 and IAP were certified with Real Time RT-PCR, and then the caspase activity in schistosome lysates was determined by using Glo? 3/7 Assay kit. Our preliminary results suggest that the SjIAP may play important roles in parasitic living and development as well as in the host–parasite interactions, and drug target of SjIAP might be a potential for controlling schistosomiasis.
4、Molecular characterizations and identification of two important genes Sj Methionine aminopeptidase 2 and Sj mago nashi those those may play important roles in growth and development of Schistosoma japonicum
Based on the result of the differentially expressed genes from the three different hosts, we selected two important functional genes Sj Methionine aminopeptidase 2 and Sj mago nashi those may play important role in growth and development of schistosoma japonicum for further research.
Genes encoding methionine aminopeptidase 2 and mago nashi of Schistosoma japonicum (Sj MAP2, Sj mago nashi) were cloned and characterized. Real time RT-PCR(reverse-transcription polymerase chain reaction) and(or) Western blot analysis revealed that SjMAP2 and Sj mago nashi were expressed by each developmental stage of the schistosome tested, but was expressed at a higher level in the schistosomulum stage and the adult male worm at 42 days. The results also showed that the two genes were differentially expressed in worms from three different host species. Sj MAP 2 and Sj mago nashi were both highly expressed in worms from the schistosome susceptible mouse, expressed at a lower level in worms from the less susceptible rat, and at an even lower level in worms from the non-permissive host Microtus fortis. The expression of Sj MAP 2 was affected significantly when the hosts were treated with praziquantel: expression was down-regulated by 92.17% and 49.01%, respectively, in treated male and female adult worms in comparison with untreated worms. An immuno-experiment in mice indicated that recombinant Sj MAP 2 and Sj mago nashi could induce partial protective efficacy against schistosome infection. The data presented here suggest that SjMAP2 and Sj mago nashi were important molecules in the growth and development of the schistosome, and that it may be a potential new drug target or vaccine candidate for schistosomiasis.
4、Molecular characterizations and identification of genes Cyclophilin A,B and C those may play important roles in signal transduction of Schistosoma japonicum
The result of the differentially expressed genes from the three different hosts revealed that the molecules as cell signaling and modification of protein translation may play important roles in the development of schistosoma japonicum. We selected Cyclophilin family genes those function may be chaperones and cell signaling as our research objects. The Cyclophilin family genes included Cyclophilin A, B and C.
The full-length cDNA encoding Cyclophilin A, B and C in Schistosoma japonicum of schistosomula were amplified by RT-PCR technique. As determined by Real-time RT-PCR, the highest expression of Sj CyPA, B and C was observed at the transcript level at schistosomula stage, indicating that Sj CyPA, B and C was a abundant expression gene in schistosomula stage.The expressions of Sj CyPA both at transprict level and at protein level were not significantly alternated upon the treatment by Cyclosporin A as determined by Real time RT-PCR and western blot. Expertly, Chymotrypsin- coupled chromogenic assay confirmed that Sj CyPA had PPIase activity. Western blot indicated that Sj CyPA, B and C were able to induce specific antibodies. Additionally, animal experiment showed that partial worm reduction and egg reduction were achieved in mice vaccinated with recombinant Sj CyPA, B and C protein, respectively.
In total, the differentially expressed genes from the three different hosts were focused on many signal pathway and function. Our research work present here demonstrated that the independentof development and growth in schistosoma japonicum from Microtus fortis may due to the nutrition metabolism, signal transduction, immune escape and apoptosis and so on. With the further research work on the differentially expressed genes, many important function molecules in Schistosolum will be characterized and its biological function will be identified. All the research work will help us to explain the host and parasite inter play, growth and development of the schistosome, and the identification of different expression genes may potentially represent new drugs or vaccine targets, applicable for the future controlling of schistosomiasis.
1、不同宿主来源日本血吸虫10d童虫形态观察及体细胞凋亡分析
实验室中以日本血吸虫尾蚴感染小鼠、大鼠和东方田鼠,10d后收集虫体。光镜下观察到不同来源日本血吸虫形态大小有很大差异,东方田鼠来源童虫长402.2±102.2μm,宽159.1±47.3μm,大鼠来源童虫长828.3±127.4μm,宽103.4±22.8μm,小鼠来源童虫长878.5±137.4μm,宽88.3±20.0μm。扫描电镜下观察,东方田鼠来源童虫虫体萎缩,口吸盘形成空泡,表面结构消失,腹吸盘表面结构改变,体表正常棘消失,萎缩,形成空泡;小鼠和大鼠来源虫体体表棘等结构无异常变化。透射电镜观察发现,东方田鼠来源童虫虫体细胞结构受到破坏,细胞崩解,细胞核仁消失,染色质凝集,有细胞凋亡的表现。小鼠和大鼠来源虫体细胞形态正常,细胞核和线粒体无明显变化。利用Annexin V-FITC/propidium iodide (PI)分析不同宿主来源虫体细胞凋亡状况表明,小鼠来源虫体的凋亡细胞比例为0.40%;大鼠来源虫体的凋亡细胞比例为5.22%;东方田鼠来源虫体的凋亡细胞比例为62.91%,表明东方田鼠来源虫体的细胞发生了明显凋亡。本研究首次利用扫描电镜和透射电镜技术,进行东方田鼠、大鼠和小鼠来源日本血吸虫童虫显微和超微结构的观察和比较,利用流式细胞仪检测发现日本血吸虫在不同宿主体内的生长发育状况与体细胞凋亡相关。
2、不同宿主来源日本血吸虫童虫基因表达的芯片分析
应用寡核苷酸芯片技术,比较分析小鼠、大鼠和东方田鼠来源的日本血吸虫10d童虫的基因表达差异。结果表明,与小鼠来源童虫相比,东方田鼠来源童虫有3293个基因呈下调表达(fold<0.5),71个基因呈上调表达(fold>2);大鼠来源童虫有3335个基因呈下调表达(fold<0.5),133个基因呈上调表达(fold>2)。其中,东方田鼠来源童虫显著性下调表达基因81个(fold<0.2),显著性上调表达基因18个(fold>5);大鼠来源童虫显著性下调表达基因210个(fold<0.2),显著性上调表达基因54个(fold>5)。东方田鼠和大鼠来源童虫具有相似表达趋势的显著性下调表达基因有27个(fold<0.2),显著性上调表达基因有7个(fold>5)。
对显著性差异表达基因进行GO分类和KEGG通路等生物信息分析,结果显示,东方田鼠来源童虫显著性上调表达基因主要与细胞(cell)、细胞凋亡(apoptosis)、细胞外组成部分(extracellular region part)、细胞组成(cell part)、酶调节活性(enzyme regulator activity)和转录调节活性(translation regulator activity)等相关。东方田鼠来源童虫显著性下调表达基因主要与代谢过程(metabolic process)、催化活性(catalytic activity)、发育过程(developmental process)、转运活性(transporter activity)、细胞进程(cellular process)、定位(localization)和结合(binding)等相关。东方田鼠和大鼠来源童虫均显著下调表达基因与代谢过程(metabolic process)、定位(localization)、结构形成自动修饰(anatomical structure formation)和催化活性(catalytic activity)等相关。对东方田鼠和大鼠来源童虫均显著下调表达的基因进行KEGG pathway通路分析,发现这些蛋白主要为甘氨酸/苏氨酸/丝氨酸代谢分子(Glycine,serine and threonine metabolism)、DNA复制关键分子(DNA replication)、MAPK信号通路分子(MAPK signaling pathway)、蛋白转运(Protein export)、氨酰基tRNA生物合成(Aminoacyl-tRNA biosythesis)等分子。本研究发现一批基因在三种宿主来源10d童虫中呈现差异表达,它们可能是不同宿主来源童虫发育差异的关键分子,影响血吸虫在不同宿主中的发育。如东方田鼠来源童虫显著上调表达,小鼠来源虫体显著下调的基因有与细胞增殖密切相关的颗粒蛋白(Granulin)基因,细胞凋亡通路中发挥凋亡效应的关键分子如Cell death protein 3,Caspase 7,caspase 3;在小鼠来源童虫显著上调表达,东方田鼠来源童虫显著下调表达的基因中与生长发育相关的甲硫氨酰氨肽酶2 (MAP 2)和Sj magonashi基因,与胰岛素代谢相关的胰岛素分子(Insulin-2)和胰岛素受体蛋白激酶(insulin receptor protein kinase)基因,与脂肪酸代谢相关的脂肪酸去饱和酶(Fatty acid desaturase)、长链脂肪酸延伸相关蛋白(Elongation of very long chain fatty acids protein)、脂肪酸脱氢酶(Fatty-acid amide hydrolase)、脂肪酸结合蛋白(fatty acid-binding protein)基因,与细胞凋亡通路中发挥抑制凋亡效应的关键分子相关Sj IAP(Baculoviral IAP repeat-containing protein)、cytokine-induced apoptosis inhibitor,与信号传导相关的Cyclophilin家族基因、TGF通路分子如Eukaryotic translation initiation factor 3,Transforming growth factor beta-1、Wnt通路分子Wnt inhibitory factor 1等。这些重要功能分子的发现,将为阐明血吸虫在不同宿主生存环境中的生长发育机制,探讨血吸虫与宿主相互作用关系、发现新的血吸虫候选疫苗分子和新药物靶标提供重要基础。
本研究分析获得了一批小鼠、大鼠和东方田鼠来源的10d童虫的差异表达基因信息,初步揭示了在不同宿主体内日本血吸虫生长发育的分子基础,发现了一批在不同宿主环境中可能影响血吸虫生长发育的关键功能分子,为这些重要差异表达基因的确定及进行生物学功能研究提供了良好的基础。
3、日本血吸虫细胞凋亡因子和凋亡抑制因子(Sj IAP)的研究
基因组水平的研究表明血吸虫与其他多细胞生物一样具有凋亡这一生物学现象。上文透射电镜研究表明,东方田鼠来源10d童虫细胞有细胞凋亡的现象,同时芯片分析结果表明,日本血吸虫凋亡相关基因caspase3和7在东方田鼠来源虫体中呈高表达,而在小鼠来源虫体中呈较低表达;日本血吸虫凋亡抑制因子基因在小鼠来源虫体中高表达,东方田鼠来源虫体中低表达,表明日本血吸虫凋亡相关基因和凋亡抑制因子可能在血吸虫生长发育和寄生虫与宿主相互作用关系中发挥重要的生物学功能,在东方田鼠抗血吸虫机制中发挥一定的作用。
本研究进行了不同宿主来源日本血吸虫童虫caspase3/7的活性和基因表达差异研究;利用RT-PCR技术获得了Sj IAP编码基因的全长cDNA,PCR扩增其mRNA对应基因组DNA序列;荧光实时定量PCR和western blot分析表明,该基因为成虫期雄虫期高表达基因;免疫组化研究分析表明,虫体IAP蛋白广泛分布于成虫体被膜;细胞水平实验证实该基因真核重组表达质粒在细胞水平可以有效抑制诱导剂引起的细胞凋亡,同时研究发现Sj IAP重组蛋白也可以在血吸虫的虫体水平抑制凋亡;Real Time RT-PCR和western blot研究了日本血吸虫凋亡抑制因子基因(IAP)在不同宿主的差异表达情况。
研究结果表明细胞凋亡相关基因很可能是不同宿主环境中影响血吸虫生长发育的重要功能分子,在血吸虫生长发育调控和东方田鼠抗血吸虫病中可能发挥重要的生物学功能。
4、日本血吸虫生长发育重要功能分子甲硫氨酰氨肽酶2基因(MAP 2)和mago nashi基因的研究
MAP 2是一种双功能酶,其催化结构域位于C端,负责切除蛋白质合成过程中新生肽链N端的起始蛋氨酸,对蛋白质的成熟以及在细胞中的转运和定位、功能调节极其重要,其N端结构域含有酸性氨基酸和碱性氨基酸富集的区域,可以抑制真核起始因子eIF2-2α的磷酸化,促进蛋白质合成的起始。在哺乳动物细胞内,MAP 2与细胞增值与分化密切相关,MAP 2在秀丽线虫生殖和发育中发挥重要的生物学功能。mago nashi基因在真核生物中广泛存在,且极为保守。在生物体翻译转录中参与mRNA定位和剪切,在生物体生殖与发育中,mago nashi基因在胚胎早期发育和生殖细胞性别决定中发挥重要的生物学功能。芯片分析结果表明,日本血吸虫MAP 2和mago nashi基因在东方田鼠来源虫体中低表达,大鼠和小鼠来源虫体中高表达。
本研究利用RT-PCR技术分别获得了Sj MAP 2(日本血吸虫甲硫氨酰氨肽酶2)和Sj mago nashi编码基因的全长cDNA(基因登录号为:GQ403663和GQ403668)。荧光实时定量PCR和western blot分析Sj MAP 2和Sj mago nashi基因在不同期别阶段虫体表达差异,发现这两个基因均为童虫期表达基因,且在雄虫的表达量均高于其在雌虫。Real Time RT-PCR和western blot分析Sj MAP 2和Sj mago nashi在不同宿主来源日本血吸虫童虫的表达情况,发现二者均在小鼠来源童虫中表达最高,大鼠次之,东方田鼠最低。应用吡喹酮处理雌虫和雄虫后,Sj M AP 2表达均有显著降低,其中雄虫和雌虫中分别下降92.17%和49.01%。构建了这2个基因的重组表达质粒,并分别在大肠杆菌中成功表达。Western blotting分析表明重组蛋白rSj MAP 2和rSj mago nashi具有良好的抗原性,应用这两种重组抗原免疫小鼠,与空白对照组比较,免疫组小鼠获得的减虫率分别为17.8%和22.43%,肝脏虫卵减少率分别为36.24%和29.11%。
以上研究表明,日本血吸虫Sj MAP 2和Sj mago nashi基因的表达可能对血吸虫生长发育有影响,这两个基因可能在血吸虫生殖、生长发育及细胞分化中具有重要的生物学功能。
5、日本血吸虫Cyclophilin家族CyPA、CyPB和CyPC基因的研究
Cyclophilin(CyP)广泛存在于生物体内,为一种分布广泛的细胞内蛋白,在植物、细菌和哺乳动物中均存在,具有高度保守性。CyP具有肽酰脯氨酰顺反异构酶(PPIase)活性,在体内外能结合蛋白质并催化加速它们的折叠、装配和转运;特别是富含脯氨酸的蛋白质折叠,起着分子伴侣的作用,同时在信号转导中起着重要调节作用。信号转导与蛋白翻译后修饰在日本血吸虫中发挥重要的生物学功能。
本研究利用RT-PCR技术分别获得了Sj CyP A、Sj CyPB和Sj CyPC编码基因的全长cDNA(基因登录号为:GQ403666、GQ403664和GQ403665)。荧光实时定量PCR分析这三个基因在不同期别阶段虫体表达量,发现这三个基因均为童虫期高表达基因。Real Time RT-PCR分别分析Sj CyP A、Sj CyPB和Sj CyPC在不同宿主来源日本血吸虫童虫的差异表达情况,发现三者均在小鼠来源童虫中表达最高,大鼠次之,东方田鼠最低。构建了这3个基因的重组表达质粒,并分别在大肠杆菌中成功表达Western blotting验证表明重组蛋白rSj CyP A、rSj CyPB和rSj CyP C均具有良好的抗原性,在小鼠免疫试验中,与空白对照组比较,三种蛋白免疫小鼠获得的减虫率分别为14.2%、26.98%和13.79%;肝脏虫卵减少率分别为43.38%、39.73%和51.32%。
研究表明,日本血吸虫Sj CyP A、Sj CyPB和Sj CyPC基因的表达可能对血吸虫生长发育有影响,这三个基因可能在血吸虫的分子伴侣、信号转导等方面发挥重要的生物学功能。
本文以日本血吸虫易感宿主BALB/c小鼠、非易感宿主Wistar大鼠和不致病宿主东方田鼠来源的10d童虫作为研究对象,应用电镜观察表明三种不同宿主来源的10d童虫在形态结构上存在较大差异。首次发现东方田鼠来源童虫虫体细胞的凋亡现象显著大于大鼠和小鼠来源虫体。首次应用寡核苷酸芯片技术对三种宿主来源童虫的差异表达基因进行了比较分析,发现一些重要的血吸虫生长发育、细胞凋亡、信号传导分子在三种不同宿主来源虫体呈差异表达,获得一批具有深入研究价值的差异表达基因信息。首次克隆、鉴定和表达了Sj MAP2, Sj magonashi, Sj CyPA, Sj CyPB和Sj CyPC五个血吸虫新基因,登录号分别为GQ403663、GQ403668 GQ403666、GQ403664和GQ403665。应用以上五种基因重组抗原进行了小鼠免疫试验,这5个基因的重组蛋白都诱导了部分免疫保护作用。首次对日本血吸虫凋亡抑制基因Sj IAP的生物学功能进行了探索,在细胞水平验证了该凋亡抑制因子可有效地抑制凋亡诱导剂诱发的凋亡。本文结果对深入探讨日本血吸虫的生长发育机制,发现血吸虫与宿主相互作用关键分子,进而为发现抗血吸虫病新的疫苗候选分子和新的治疗药物靶标提供了新思路。
Schistosomiasis is one of the wide spread and prevalent parasitic diseases worldwide. Nearly 40 kinds of animals can be infected by Schistosoma japonicum (Chinese mainland strain). The previous research had demonstrated that BALB/c mouse as a susceptible host of schistosome, Wistar rat as a unsusceptible host and Microtus fortis as resistant host of Schistosoma japonicum infection. Microtus fortis is the resistant host of Schistosoma japonicum. Animals have different symptoms after infected by cercaria: 60-70% of the adult Schistosoma japonicum can be perfused post-cercarial challenge from the host in BALB/c mouse, but 10-20% from Wistar Rat. Most of the schistosolums have incomplete development and fail to accomplish their life cycles in Microtus fortis. Till now, Microtus fortis is the only unmorbific host after infected with schistosoma japonicum. The microarray analysis of the gene expression difference between mouse, Wistar rat and Microtus fortis demonstrated that the genes related with immune mechanism, apoptosis, growth and development were differential expressed. Our research work focused on the different expression genes in 10 days Schistosolum from the hosts of Microtus fortis, BALB/c mouse and Wistar rat with the analysis of oligonucleotide microarray and bioinformatics techniques. A great number of genes related with signal transduction, immune escape, development and metabolism had been screen out with the analysis. Several genes of Schistosoma japonicum were selected for further research: genes referred with apoptosis such as Sj IAP、caspase3 and caspase 7, genes related with development and growth such as Sj MAP 2 and Sj mago nashi, in signal transduction such as Cyclophilin A,B and C genes.Our data presented here suggest that such genes may play important role in the development; reproduction and host-parasite interplay in schistosome living, and may be a potential new drugs or vaccine target for controlling schistosomiasis.
1、Scanning electron microscope(SEM) and transmission electron microscopy (TEM) observation of Schistosolum from the three different hosts
There are obvious changes in the development and growth of Schistosoma japonicum in different hosts. After two weeks post infected with cercaria, Schistosolum gradually withered and stopped growth, then died. The width and length of schistosomula were measured with optical microscope.The length of schistosomula from Microtus fortis, Wistar rat and BALB/c mouse was 402.2±102.2, 828.3±127.4 and 878.5±137.4. The width of schistosomula from Microtus fortis, Wistar rat and BALB/c mouse was 159.1±47.3,103.4±22.8 and 88.3±20.0.The morphologic comparion of the schistosolums from three different hosts with SEM and TEM showed that microscopic and ultrastructural structure of the schistosolums have significant difference. The surfaces of the schistosomula from Microtus fortis were stunted, shrinking and vacuole.The nucleus of the cells in schistosomula from Microtus fortis were agglutinated. All these revealed that the cells of Schistosolum may have the phenomenon of apoptosis. Schistosolum were divided into monoplast with trypsinization, and then apoptosis were determined by flow cytometry (FCM) with the analysis of Annexin V-FITC/propidium iodide (PI).
2、Analyze differentially expressed genes of 10 days schistosolum from the three different hosts
Based on the data of oligonucleotide microarray and bioinformatics analysis of the differentially expressed genes from the three different hosts: in the schistosolums from Microtus fortis,3293 genes were down-regulated (fold <0.5)and 71 genes were up-regulated (fold >2) in comparison with the schistosolums in BALB/c mouse; in the schistosolums in from Wistar rat , 3335 geneswere down-regulated and 133 genes were up-regulated in comparison with the schistosolums in BALB/c mouse.
Preliminary function analysis with Gene Ontology GO classification and KEGG pathways analysis indicate that the up-regulated expressed genes in the schistosolums from Microtus fortis focused on the function as cell, extracellular region part, cell part, enzyme regulator activity, translation regulator activity, the down-regulated expressed genes in the schistosolums from Microtus fortis focused on the function as metabolic process, catalytic activity, developmental process, transporter activity, cellular process, localization and binding. The significant down-regulated expressed genes (fold <0.2) in the schistosolums from both Microtus fortis and Wistar rat included metabolic process, localization, anatomical structure formation and catalytic activity. Also another some genes not related with the above process are also co-down regulated, such as serine/threonine-protein kinase, NAD(P)H-quinone oxidoreductase, Pericentriolar material, Cell surface receptor daf-4. The KEGG pathway analysis revealed that the co-down regulated expressed genes in the schistosolums from both Microtus fortis and Wistar rat consist of the pathway of Glycine, serine and threonine metabolism, DNA replication, MAPK signaling pathway, Protein export, Aminoacyl-tRNA biosythesis. Because the genomic research works of S. japonicum and S. mansoni were fall behind the Model organism Caenorhabditis elegans, we do the KEGG pathway analysis of the co-down regulated expressed genes with the C. elegans pathway model. We found that some genes are involved in the pathway as Apoptosis, Oxidative phosphorylation, Insulin signaling pathway and Fatty acid metabolism and so on.
The 10 days schistosolum specific expression genes were selected and analyzed with the gene expression of NADH dehydrogenase [ubiquinone] flavoprotein 2 as the internal control. The result showed that 1178 genes were down-regulated (fold <0.5) and 524 genes were up-regulated (fold >2), also 124 significant down-regulated genes (fold <0.2) and 578 significant up-regulated genes (fold >5). The result of the Gene Ontology GO classification analysis of the significant up-regulated/high genes demonstrated that the different function genes between these two trend genes focused on metabolic process, catalytic activity, enzyme regulator activity, transcription regulator activity, transporter activity, antioxidant activity, electron carrier activity, structural molecule activity. In the other part, the KEGG pathways of the different function genes are involved in Protein export, MAPK signaling pathway, Oxidative phosphorylation, Inositol phosphate metabolism, Calcium signaling pathway, Progesterone-mediated oocyte maturation), Ubiquitin mediated proteolysis, Notch signaling pathway and estrogen-related receptor beta like 2.
3、Analyzed of apoptosis in Schistosoma japonicum from different hosts and molecular characterizations of an inhibitor of apoptosis
Apoptosis is a normal process for regulating cellular death of many organisms. We molecularly characterized an inhibitor of apoptosis from Schistosoma japonicum (SjIAP). The transcription of the SjIAP predominantly occurred at the developmental stages in a final host. The immunohistochemistry analysis revealed that Sj IAP protein was located in the tegument of the adult male worm of schistosoma japonicum. Functional assay indicated that the SjIAP could inhibit caspase activity either in 293T cell or in schistosome lysates. Additionally, there were differently expressed profiles of the SjIAP in S. japonicum living in different hosts. The gene expression of two important molecules caspase 3 caspase 7 and IAP were certified with Real Time RT-PCR, and then the caspase activity in schistosome lysates was determined by using Glo? 3/7 Assay kit. Our preliminary results suggest that the SjIAP may play important roles in parasitic living and development as well as in the host–parasite interactions, and drug target of SjIAP might be a potential for controlling schistosomiasis.
4、Molecular characterizations and identification of two important genes Sj Methionine aminopeptidase 2 and Sj mago nashi those those may play important roles in growth and development of Schistosoma japonicum
Based on the result of the differentially expressed genes from the three different hosts, we selected two important functional genes Sj Methionine aminopeptidase 2 and Sj mago nashi those may play important role in growth and development of schistosoma japonicum for further research.
Genes encoding methionine aminopeptidase 2 and mago nashi of Schistosoma japonicum (Sj MAP2, Sj mago nashi) were cloned and characterized. Real time RT-PCR(reverse-transcription polymerase chain reaction) and(or) Western blot analysis revealed that SjMAP2 and Sj mago nashi were expressed by each developmental stage of the schistosome tested, but was expressed at a higher level in the schistosomulum stage and the adult male worm at 42 days. The results also showed that the two genes were differentially expressed in worms from three different host species. Sj MAP 2 and Sj mago nashi were both highly expressed in worms from the schistosome susceptible mouse, expressed at a lower level in worms from the less susceptible rat, and at an even lower level in worms from the non-permissive host Microtus fortis. The expression of Sj MAP 2 was affected significantly when the hosts were treated with praziquantel: expression was down-regulated by 92.17% and 49.01%, respectively, in treated male and female adult worms in comparison with untreated worms. An immuno-experiment in mice indicated that recombinant Sj MAP 2 and Sj mago nashi could induce partial protective efficacy against schistosome infection. The data presented here suggest that SjMAP2 and Sj mago nashi were important molecules in the growth and development of the schistosome, and that it may be a potential new drug target or vaccine candidate for schistosomiasis.
4、Molecular characterizations and identification of genes Cyclophilin A,B and C those may play important roles in signal transduction of Schistosoma japonicum
The result of the differentially expressed genes from the three different hosts revealed that the molecules as cell signaling and modification of protein translation may play important roles in the development of schistosoma japonicum. We selected Cyclophilin family genes those function may be chaperones and cell signaling as our research objects. The Cyclophilin family genes included Cyclophilin A, B and C.
The full-length cDNA encoding Cyclophilin A, B and C in Schistosoma japonicum of schistosomula were amplified by RT-PCR technique. As determined by Real-time RT-PCR, the highest expression of Sj CyPA, B and C was observed at the transcript level at schistosomula stage, indicating that Sj CyPA, B and C was a abundant expression gene in schistosomula stage.The expressions of Sj CyPA both at transprict level and at protein level were not significantly alternated upon the treatment by Cyclosporin A as determined by Real time RT-PCR and western blot. Expertly, Chymotrypsin- coupled chromogenic assay confirmed that Sj CyPA had PPIase activity. Western blot indicated that Sj CyPA, B and C were able to induce specific antibodies. Additionally, animal experiment showed that partial worm reduction and egg reduction were achieved in mice vaccinated with recombinant Sj CyPA, B and C protein, respectively.
In total, the differentially expressed genes from the three different hosts were focused on many signal pathway and function. Our research work present here demonstrated that the independentof development and growth in schistosoma japonicum from Microtus fortis may due to the nutrition metabolism, signal transduction, immune escape and apoptosis and so on. With the further research work on the differentially expressed genes, many important function molecules in Schistosolum will be characterized and its biological function will be identified. All the research work will help us to explain the host and parasite inter play, growth and development of the schistosome, and the identification of different expression genes may potentially represent new drugs or vaccine targets, applicable for the future controlling of schistosomiasis.
引文
1.程国锋.日本血吸虫性别差异蛋白质组及抱雌沟蛋白基因功能研究[博士论文].北京:中国农科院研究生院,2004.
2.周述龙,林建银,蒋明森.血吸虫学[M].第2版.北京:科学出版社,2001.
3.鲍世民,沈志明,余家磺.东方田鼠乳酸脱氢酶同工酶研究[J].上海实验动物科学,1994, 14(34): 175~178.
4.陈利玉,易新元,曾宪芳,等.日本血吸虫新基因SjF1的克隆、表达及免疫保护效果研究[J].中国人兽共患病杂志,2004,20(6):467~470.
5.傅志强,刘金明,蒋守富,等.东方田鼠抗日本血吸虫抗体水平监测与分析[J].中国兽医寄生虫病,2001,9(4):6~8.
6.韩海勃,曹建平,刘述先.日本血吸虫硫氧还蛋白编码基因的克隆和表达[J].中国人兽共患病杂志,2005,(12):175~178
7.韩海勃,曹建平,刘述先,等.日本血吸虫重组硫氧还蛋白小鼠保护性免疫研究[J].中国寄生虫学与寄生虫病杂志,2006,(03):17~19
8.蒋守富,魏梅雄,林矫矫,等.东方田鼠抗日本血吸虫抗体IgG亚类的初步研究[J].中国血吸虫病防治杂志,2001,13(1):1~3
9.蒋守富,魏梅雄,林矫矫,等.东方田鼠血清被动转移抗日本血吸虫的保护力研究[J].中国血吸虫病防治杂志,2004,17(5):298~230.
10.黎申恺,朱祖林,金壁如,等.东方田鼠对日本血吸虫的不感染性[J].寄生虫学报,1965,2(1):103~105
11.李浩,何艳燕,林矫矫,等.东方田鼠抗日本血吸虫病现象的观察[J].中国兽医寄生虫病,2000,8(2):12~15.
12.李浩,何艳燕,林邦发,等.东方田鼠重复感染日本血吸虫试验研究初步[J].中国兽医寄生虫病2001, 9(3):15~17
13.刘金明,傅志强,李浩,等.东方田鼠ADCC体外杀伤日本血吸虫童虫效果的初步观察[J].寄生虫与医学昆虫学报,2001,8(4):212~219.
14.刘金明,傅志强,李浩,等.东方田鼠血清体外杀伤日本血吸虫童虫效果的初步观察[J].中国人兽共患病杂志,2002,18(2):82~84.
15.刘庆中,沈继龙.日本血吸虫重组信号蛋白14-3-3疫苗免疫保护作用的观察[J].中国人兽共患病杂志,2004,20(3):230~232
16.罗新松,何永康,喻鑫玲,等.东方田鼠血清被动转移小白鼠抗血吸虫感染的保护性研究[J].中国人兽共患病杂志,1998,14(5),75~76
17.何永康,罗新松,张新跃,等.东方田鼠天然抗血吸虫感染免疫特性的研究[J].中国血吸虫病防治杂志,1998,10(增刊):73~77
18.何永康,宋光承,刘述先,等.重组日本血吸虫26kDa GST抗原免疫水牛后抗体动态及免疫保护性效果[J].中国寄生虫学与寄生虫病杂志,2001,19(5):265~267
19.何永康,刘述先,喻鑫玲,等.重组日本血吸虫26kDa GST抗原诱导水牛产生抗体及减少排卵的初步观察[J].中国寄生虫学与寄生虫病杂志,2002,20(3):133~135
20.缪应新,刘述先.日本血吸虫磷酸丙糖异构酶小鼠免疫试验[J].中国寄生虫学与寄生虫病杂志,1996,14(4):257~261
21.欧阳理,易新元,曾宪芳,等.东方田鼠血清及其不同部分对日本血吸虫童虫的体外杀伤作用[J].中国血吸虫病防治杂志,2003,15:98~102.
22.汪世平,易新元,曾宪芳,等.东方田鼠血清体外杀童虫效果的初步观察[J].中国人兽共患病杂志,1994,5(1):188~190.
23.王庆林,易新元,罗新松,等.日本血吸虫肠道蛋白酶对小鼠和东方田鼠血红蛋白降解作用的比较[J].中国人兽共患病杂志,2000,16(4):35~36.
24.王庆林,易新元,曾宪芳,等.东方田鼠感染日本血吸虫后肺和肝的组织细胞反应及虫体的扫描电镜观察[J].中国人兽共患病杂志,2002,18(4):65~67.
25.王庆林,易新元,曾宪芳,等.东方田鼠感染日本血吸虫后肺和肝的组织细胞反应及虫体的扫描电镜观察[J].中国人兽共患病杂志,2002, 18(4): 65~67.
26.王陇德,周晓农,陈红根,等.血吸虫病控制新策略的研究[J].中国工程科学,2009,11(5):37~43.
27.邬国军,秦志强,罗赛群,等.东方田鼠血清组分对日本血吸虫童虫的体外杀伤力[J].中国寄生虫学与寄生虫病杂志,2005,23(6):404~407.
28.吴光,张容,杨元清,等.日本血吸虫实验感染东方田鼠的初步报告[J].中国医学科学院寄生虫病研究所年报,1962:189~190.
29.张新跃,何永康,李毅,等.正常东方田鼠血清及脾细胞体外杀血吸虫童虫作用的初步观察[J].中国血吸虫病防治杂志,2001,13:206~208.
30.朱国正,汪英华,雷观愚,等.东方田鼠的实验室饲养及其抗血吸虫感染特性[J].上海实验动物科学,1991,11(4):193~198
31. Albuquerque SS, Carret C, Grosso AR, et al. Host cell transcriptional profiling during malaria liver stage infection reveals a coordinated and sequential set of biological events[J].BMC Genomics. 2009.10:270.
32. Arévalo-Rodríguez M, Heitman J. Cyclophilin A is localized to the nucleus and controls meiosis in Saccharomyces cerevisiae[J]. Eukaryotic Cell. 2005.4(1):17-29
33. Bornmann WG, Sirotnak FM, Scheinberg DA.Human mitochondrial peptide deformylase, a new anticancer target of actinonin-based antibiotics[J]. J Clin Invest.2004 114(8):1107-16.
34. Boxem M, Tsai CW, Zhang Y, et al. The C. elegans methionine aminopeptidase 2 analog map-2 is required for germ cellproliferation[J].FEBS Lett. 2004.576(1-2):245-50.
35. Bugli F, Khattab A, Vigneti E, et al. Expression cloning and biochemical characterizations of recombinant Cyclophilin proteins from Schistosoma mansoni[J]. Protein Expression Purification. 1998.12(3):340-6.
36. Capron A, Dessaint JP. Molecular basis of host–parasite relationship: towards the definition ofprotective antigens[J]. Immunol Rev.1989.112:27-48
37. Caffrey CR, Gsell C, Ruppel A. Schistosoma japonicum is less sensitive to cyclosporin A in vivo than Schistosoma mansoni[J].Journal of Parasitology. 1999.85(4):736-9
38. Chai SC, Ye QZ.Metal-mediated inhibition is a viable approach for inhibiting cellular methionine aminopeptidase[J].Bioorg Med Chem Lett .2009.19(24):6862-4
39. Chen L, Rao KV, He YX, Ramaswamy K.Skin-stage schistosomula of Schistosoma mansoni produce an apoptosisinducing factor that can cause apoptosis of T cells[J].J Biol Chem. 2002.277(37):34329-34335
40. Cheng G, Davis RE.An improved and secreted luciferase reporter for schistosomes[J]. Mol Biochem Parasitol .2007.155(2):167-171
41. Cheng GF, Lin JJ, Feng XG, et al. Proteomic analysis of differentially expressed proteins between the male and female worm of Schistosoma japonicum after pairing[J].Proteomics. 2005.5(2):511–521
42. Cheng G, Fu Z, Lin J, et al. In vitro and in vivo evaluation of small interference RNA-mediated gynaecophoral canal protein silencing in Schistosoma japonicum[J].J Gene Med .2009.11(5):412-421
43. Chen X, Xie S, Bhat S, Kumar N, et al. Fumagillin and fumarranol interact with P. falciparum methionine aminopeptidase 2 and inhibit malaria parasite growth in vitro and in vivo[J].Chem Biol .2009.16(2):193-202
44. Chu FH, Chen YR, Lee CH, et al. Molecular characterization and expression analysis of Acmago and AcY14 in Antrodia cinnamomea[J].Mycol Res, 2009.113(5): 577-582.
45. Clare S. Gavigana, Senan P. Kielya, Jocelyne Hirtzlinb, et al. Cyclosporin-binding proteins of Plasmodium falciparum[J].International Journal for Parasitology.2003. 33:987-996
46. Cutforth T, Gaul U.A methionine aminopeptidase and putative regulator of translation initiation is required for cell growth and patterning in Drosophila[J].Mech Dev.1999.82(1-2):23-8
47. Degot S, Le Hir H, Alpy F, et al. Tomasetto C.Association of the breast cancer protein MLN51 with the exon junction complex via its speckle localizer and RNA binding module[J]. J Biol Chem, 2004, 79(32):33702-15.
48. Dewey WC, Ling CC, Meyn RE. Radiation-induced apoptosis: relevance to radiotherapy[J]. Int J Radiat Oncol Biol Phys .1995.33 (4):781-796
49. Ettinger NA, Wilson ME. Macrophage and T-cell gene expression in a model of early infection with the protozoan Leishmania chagasi[J]. PLoS Negl Trop Dis. 2008. 25;2(6):e252.
50. Evans H, Mello LV, Fang Y, et al. Microarray analysis of gender- and parasite-specific gene transcription in Strongyloides ratti[J].Int J Parasitol. 2008.38(11):1329-41.
51. Friedrich N, Santos JM, Liu Y, et al. Soldati-Members of a novel protein family containing microneme adhesive repeat domains act as sialic acid-binding lectins during host cell invasion by apicomplexan parasites[J].J Biol Chem. 2010.285(3):2064-76.
52. Gobert GN, McManus DP, Nawaratna S, et al. Tissue specific profiling of females of Schistosoma japonicum by integrated laser microdissection microscopy and microarray analysis[J].PoS Negl Trop Dis. 2009 .30;3(6):e469.
53. Gobert GN, Moertel L, Brindley PJ, et al. Developmental gene expression profiles of the human pathogen Schistosoma japonicum[J].BMC Genomics. 2009.25;10:128.
54. Gupta BC.Basch PF. The role of Schistosoma mansoni males in feeding and development of female worms[J]. J Parasitology. 1987.73(3):674.
55. Hachet O, Ephrussi A. Drosophila Y14 shuttles to the posterior of the oocyte and is required for oskar mRNA transport[J]. Curr Biol, 2001, 11(21):1666-74.
56. Hachet O, Ephrussi A. Splicing of oskar RNA in the nucleus is coupled to its cytoplasmic localization[J]. Nature, 2004, 428(6986):959-63.
57. Henkle KJ,Cook JA,Foster LA, et al. The gene family encoding eggshell proteins of Schistosoma japonicum[J].Vaccine,1997,15(8):846-848.
58. Horibe T, Yosho C, Okada S, et al. The chaperone activity of protein disulfide isomerase is affected by cyclophilin B and cyclosporin A in vitro[J]. J .Bio chem. 2002;132(3):401-7.
59. Hu X,,Addlagatta A, Lu J, et al. Elucidation of the function of type 1 human methionine aminopeptidase during cell cycle progression[J].Proc Natl Acad Sci U S A 2006.103(48):18148-53
60. Khattab A, Pica-Mattoccia L, Klinkert MQ, et al. Cyclosporins: lack of correlation between antischistosomal properties and inhibition of cyclophilin isomerase activity[J]. Experimental Parasitology. 1998 .90(1):103-9.
61. Kim DM, Ko BS, Ju JW, et al. Gene expression profiling in mouse liver infected with Clonorchis sinensis metacercariae[J].Parasitol Res. 2009 .106(1):269-78
62. Klinkert MQ, Bugli F, Cruz J, et al. Sequence conservation of schistosome cyclophilins[J]. Molecular & Biochemical Parasitology. 1996.30;81(2):239-42
63. Kumar S, Tamura K, Nei M () MEGA3: integrated software for Molecular Evolutionary Genetics. Analysis and sequence alignment[J].Brief Bioinform .2004.5(2):150-163
64. Lee DY, Seto P, Korczak R.DNA microarray-based detection and identification of waterborne protozoan athogens[J]. Microbiol Methods. 2010.80(2):129-33
65. Li BW, Rush AC, Mitreva M, et al. Transcriptomes and pathways associated with infectivity, survival and immunogenicity in Brugia malayi L3[J].BMC Genomics. 2009.15;10:267.
66. Li W, Boswell R, Wood WB. mag-1, a homolog of Drosophila mago nashi, regulates hermaphrodite germ-line sex determination in Caenorhabditis elegans[J]. Dev Biol, 2000, 218(2):172-82.
67. Liu F, Zhou Y, Wang ZQ, et al. The Schistosoma japonicum genome reveals features of host-parasite interplay[J].Nature. 2009.460(7253):345-51.
68. Liu SX, Yongkang H, Guangchen S, et al. Anti-fecundiimmunity to Sohistosoma japonicum induced in Chinese water buffaloes (Bos buffelus) after vaccination with recombinant 26 kDaglutathione -S -transferase(reSjc26GST) [J].Vet Parasitol, 1997.69:39-47.
69. Marfurt J, Smith TA, Hastings IM, et al. Plasmodium falciparum resistance to anti-malarial drugs in Papua New Guinea:evaluation of a community-based approach for the molecular monitoring of resistance[J].Malar J. 2010 .7;9:8.
70. McManus DP,Wong JY,Zhou J, et al. Recombinant paramyosin (rec-Sj-97) tested for immunogenicity and vaccine efficacy against Schistosomajaponicum in mice and water buffaloes[J].Vaccine,2001.20(5-6):870-878.
71. Minning TA, Weatherly DB,et al. The steady-state transcriptome of the four major life-cycle stages of Trypanosoma cruzi[J].BMC Genomics. 2009 .7;10:370.
72. Michael J. Smout, Thewarach Laha,Jason Mulvenna, et al. Granulin-Like Growth Factor Secreted by the Carcinogenic Liver Fluke, Opisthorchis viverrini, Promotes Proliferation of Host Cells[J].PLoS Pathogens,2009,5(10 ) e1000611,1-16
73. Mohr SE, Dillon ST, Boswell RE. The RNA-binding protein Tsunagi interacts with Mago Nashi to establish polarity and localize oskar mRNA during Drosophila oogenesis[J]. Genes Dev, 2001, 15(21):2886-99.
74. 0hta N,Kumagai T,Maruyama H, et al. Research on calpain of Schistosoma japonicum as a vaccine candidate[J].Parasitol lnt.2004.53(2):l75-181.
75. Parma DH, Bennett PE Jr, Boswell RE. Mago Nashi and Tsunagi/Y14, respectively, regulate Drosophila germline stem cell differentiation and oocyte specification[J].Dev Biol, 2007, 308(2):507-19.
76. Park NI, Muench DG. Biochemical and cellular characterization of the plant ortholog of PYM, a protein that interacts with the exon junction complex core proteins Mago and Y14[J].Planta, 2007, 225(3):625-39.
77. Roger Pelle′a, Francis McOdimbaa, Francis Chumaa, et al. The African trypanosome cyclophilin A homologue contains unusual conserved central and N-terminal domains and is developmentally regulated[J].Gene,2002.290:181-191
78. Richter D, Harn DA, Matuschka FR. The irradiated cercariae vaccine model: looking on the bright side of radiation[J].Parasitol Today.1995.11(8):288-293
79. Rycyzyn MA, Reilly SC, O'Malley K, et al. Role of cyclophilin B in prolactin signal transduction and nuclear retrotranslocation[J]. Mol Endocrinol. 2000;4(8):1175-86.
80. Salzet M, Capron A, Stefano GB. Molecular crosstalk in host– parasite relationships: schistosome– and leech–host interactions[J].Parasitol Today.2000.6(12):536-540
81. Sean Dobson,Takiko May,Matthew Berriman, et al. Characterization of protein Ser:Thr phosphatases of the malaria parasite, Plasmodium falciparum: inhibition of the parasitic calcineurin by cyclophilin-cyclosporin complex[J].Molecular and Biochemical Parasitology.1999.167-181
82. Serra EC, Lardans V, Dissous C.Identification of NF-AT-like transcription factor in Schistosoma mansoni: its possible involvement in the antiparasitic action of cyclosporin A[J].Molecular & Biochemical Parasitology. 1999.25;101(1-2):33-41.
83. Sherry, B., Yarlett, N., Strupp, A., et al. Identification of cyclophilin as a proinflammatory secretory product of lipopolysaccharide-activated macrophages[J]. Proc Natl Acad Sci U S A. 1992.89(8):3511-5.
84. Scott JC,McManus DE. Identification of novel 70-kDa heat shock protein-encoding cDNAs from Schistosoma japonicum[J].Int J Parasitol.1999.29(3):437-444.
85. Sugiyama H,Kawanaka M,Kameoka Y, et al. A novel cDNA clone of Schutosoma japonicum encoding the 34,000 Dalton proteins of Schistosoma japonicum[J].Mol Biochem Parasitol,1990.42(1):69-82.
86. Swidzinski JA, Zaplachinski ST, Chuong SD, et al. Molecular characterization and expression analysis of a highly conserved rice mago nashil homolog[J]. Genome, 2001, 44(3):394-400.
87. Salvesen GS, Duckett CS. IAP proteins: blocking the road to death’s door[J].Nat Rev Mol Cell Biol .2002.3(6):401-410
88. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[J].Nucleic Acids Res .1994.22 (22):4673-4680
89. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science .1998.281(5381):1312-1316
90. Upadhya R, Zhang HS, Weiss LM () System for expression of microsporidian methionine amino peptidase type 2(MetAP2) in the yeast Saccharomyces cerevisiae[J].Antimicrob Agents Chemother .2006.50(10):3389-95
91. Van der Weele CM, Tsai CW, Wolniak SM. Mago nashi is essential for spermatogenesis in Marsilea[J]. Mol Biol Cell, 2007.18(10):3711-22.
92. Vicogne J, Cailliau K, Tulasne D, et al. Conservation of epidermal growth factor receptor function in the human parasitic helminth Schistosoma mansoni[J].J Biol Chem.2004. 279(36):37407-37414
93. Veitch NJ, Johnson PC, Trivedi U, et al. Digital gene expression analysis of two life cycle stages of the human-infective parasite, Trypanosoma brucei gambiense reveals differentially expressed clusters of co-regulated genes[J]. BMC Genomics. 2010. 22;11:124.
94. Verity C,McManus DP,Brindley PJ. Vaccine efficacy of recombinant cathepsin D aspartic protease from Sohistosoma japonicum.Parasite lmmunol.2001.23:153-162.
95. Vyacheslav Yurchenko,Zhu Xue,Barbara Sherry, et al. Functional analysis of Leishmania major cyclophilin[J],International Journal for Parasitology 2008.(38):633-639
96. Wang P, Heitman J.The cyclophilins[J].Genome Biol. 2005.6(7):226..
97. Waine GJ,Yang W,Scott JC, et al. DNA-based vaccination using Schistosoma japonicum (Asian blood—fluke)genes eggshell precursor protein[J].Int J Parasitol.1997.27(7):8ll-8l7.
98. Wang L, Utzinger J, Zhou XN. Schistosomiasis control:experiences and lessons from China[J].Lancet .2008.372(9652):1793-1795
99. Wang X, Gobert GN, Feng X,et al. Analysis of early hepatic stage schistosomula geneexpression by subtractive expressed sequence tags library[J].Mol Biochem Parasitol. 2009.166(1):62-9
100.Yang YL, Serrano MG, Sheoran AS, et al. Over-expression and localization of a host protein on the membrane of Cryptosporidium parvum infected epithelial cells[J].Mol Biochem Parasitol. 2009 Nov;168(1):95-101
101.Yoshino TP, Boyle JP, Humphries JE. Receptor-ligand interactions and cellular signalling at the host–parasite interface[J].Parasitology. 2001.123.l:S143-S157
102.Yu F, Li Y, Liu L. Comparative genomics of human-like Schistosoma japonicum genes indicates a putative mechanism for host-parasite relationship[J].Genomics. 2008.91(2):152-157
103.You H, Zhang W, Moertel L, et al. Transcriptional profiles of adult male and female Schistosoma japonicum in response to insulin reveal increased expression of genes involved in growth and development[J].Int J Parasitol. 2009 .39(14):1551-9.
104.Yurchenko, V., Zybarth, G., O’Connor,M., , et al. Active site residues of cyclophilinA are crucial for its signaling activity via CD147 [J].Journal of Biological Chemistry. 2002.277.22959-22965.
105.Yurchenko, V., Constant, S., Bukrinsky, M., Dealing with the family:CD147 interactions with cyclophilins[J].Immunology 2006.117, 301-309.
106.Zeiner GM, Norman KL, Thomson JM,, et al. Toxoplasma gondii infection specifically increases the levels of key host microRNAs[J].PLoS ONE.2010.5(1 )e8742
107.Zhang YY,Taylor MG,Gregoriadis G, et al. Immunogenicity of plaslnid DNA encoding the 62 kDa fragment of Schistosoma iaponicum mysin [J].Vaccine.2000.18(20):2102-2109
108.Zhu C, Wang X, Deinum J, et al. Cyclophilin A participates in the nuclear translocation of apoptosis-inducing factor in neurons after cerebral hypoxia-ischemia[J].Journal of Experimental Medicine. 2007.204(8):1741-45
109.Zhu Y,Ren J,Ham DA, et al. Protective immunity induced with 23 kDa membrane protein DNA vaccine of Schistosoma japonicum Chinese strain in infected C57BL / 6 mice[J].Southeast Asian Trop Med Pub Hehh.2003.34(4):697-701.
110.Zhou Y, Zheng H, Liu F, et al. The Schistosoma japonicum genome reveals features of host–parasite interplay[J].Nature .2009.460(7253):345-351
111.Zeng T, Cai L, Zeng Q, et al. Immunization of mice with cells from juvenile worms of Schistosoma japonicum provides immunoprotection against schistosomiasis[J].Sci China C Life Sci. 2007.50(6):822-30
2.周述龙,林建银,蒋明森.血吸虫学[M].第2版.北京:科学出版社,2001.
3.鲍世民,沈志明,余家磺.东方田鼠乳酸脱氢酶同工酶研究[J].上海实验动物科学,1994, 14(34): 175~178.
4.陈利玉,易新元,曾宪芳,等.日本血吸虫新基因SjF1的克隆、表达及免疫保护效果研究[J].中国人兽共患病杂志,2004,20(6):467~470.
5.傅志强,刘金明,蒋守富,等.东方田鼠抗日本血吸虫抗体水平监测与分析[J].中国兽医寄生虫病,2001,9(4):6~8.
6.韩海勃,曹建平,刘述先.日本血吸虫硫氧还蛋白编码基因的克隆和表达[J].中国人兽共患病杂志,2005,(12):175~178
7.韩海勃,曹建平,刘述先,等.日本血吸虫重组硫氧还蛋白小鼠保护性免疫研究[J].中国寄生虫学与寄生虫病杂志,2006,(03):17~19
8.蒋守富,魏梅雄,林矫矫,等.东方田鼠抗日本血吸虫抗体IgG亚类的初步研究[J].中国血吸虫病防治杂志,2001,13(1):1~3
9.蒋守富,魏梅雄,林矫矫,等.东方田鼠血清被动转移抗日本血吸虫的保护力研究[J].中国血吸虫病防治杂志,2004,17(5):298~230.
10.黎申恺,朱祖林,金壁如,等.东方田鼠对日本血吸虫的不感染性[J].寄生虫学报,1965,2(1):103~105
11.李浩,何艳燕,林矫矫,等.东方田鼠抗日本血吸虫病现象的观察[J].中国兽医寄生虫病,2000,8(2):12~15.
12.李浩,何艳燕,林邦发,等.东方田鼠重复感染日本血吸虫试验研究初步[J].中国兽医寄生虫病2001, 9(3):15~17
13.刘金明,傅志强,李浩,等.东方田鼠ADCC体外杀伤日本血吸虫童虫效果的初步观察[J].寄生虫与医学昆虫学报,2001,8(4):212~219.
14.刘金明,傅志强,李浩,等.东方田鼠血清体外杀伤日本血吸虫童虫效果的初步观察[J].中国人兽共患病杂志,2002,18(2):82~84.
15.刘庆中,沈继龙.日本血吸虫重组信号蛋白14-3-3疫苗免疫保护作用的观察[J].中国人兽共患病杂志,2004,20(3):230~232
16.罗新松,何永康,喻鑫玲,等.东方田鼠血清被动转移小白鼠抗血吸虫感染的保护性研究[J].中国人兽共患病杂志,1998,14(5),75~76
17.何永康,罗新松,张新跃,等.东方田鼠天然抗血吸虫感染免疫特性的研究[J].中国血吸虫病防治杂志,1998,10(增刊):73~77
18.何永康,宋光承,刘述先,等.重组日本血吸虫26kDa GST抗原免疫水牛后抗体动态及免疫保护性效果[J].中国寄生虫学与寄生虫病杂志,2001,19(5):265~267
19.何永康,刘述先,喻鑫玲,等.重组日本血吸虫26kDa GST抗原诱导水牛产生抗体及减少排卵的初步观察[J].中国寄生虫学与寄生虫病杂志,2002,20(3):133~135
20.缪应新,刘述先.日本血吸虫磷酸丙糖异构酶小鼠免疫试验[J].中国寄生虫学与寄生虫病杂志,1996,14(4):257~261
21.欧阳理,易新元,曾宪芳,等.东方田鼠血清及其不同部分对日本血吸虫童虫的体外杀伤作用[J].中国血吸虫病防治杂志,2003,15:98~102.
22.汪世平,易新元,曾宪芳,等.东方田鼠血清体外杀童虫效果的初步观察[J].中国人兽共患病杂志,1994,5(1):188~190.
23.王庆林,易新元,罗新松,等.日本血吸虫肠道蛋白酶对小鼠和东方田鼠血红蛋白降解作用的比较[J].中国人兽共患病杂志,2000,16(4):35~36.
24.王庆林,易新元,曾宪芳,等.东方田鼠感染日本血吸虫后肺和肝的组织细胞反应及虫体的扫描电镜观察[J].中国人兽共患病杂志,2002,18(4):65~67.
25.王庆林,易新元,曾宪芳,等.东方田鼠感染日本血吸虫后肺和肝的组织细胞反应及虫体的扫描电镜观察[J].中国人兽共患病杂志,2002, 18(4): 65~67.
26.王陇德,周晓农,陈红根,等.血吸虫病控制新策略的研究[J].中国工程科学,2009,11(5):37~43.
27.邬国军,秦志强,罗赛群,等.东方田鼠血清组分对日本血吸虫童虫的体外杀伤力[J].中国寄生虫学与寄生虫病杂志,2005,23(6):404~407.
28.吴光,张容,杨元清,等.日本血吸虫实验感染东方田鼠的初步报告[J].中国医学科学院寄生虫病研究所年报,1962:189~190.
29.张新跃,何永康,李毅,等.正常东方田鼠血清及脾细胞体外杀血吸虫童虫作用的初步观察[J].中国血吸虫病防治杂志,2001,13:206~208.
30.朱国正,汪英华,雷观愚,等.东方田鼠的实验室饲养及其抗血吸虫感染特性[J].上海实验动物科学,1991,11(4):193~198
31. Albuquerque SS, Carret C, Grosso AR, et al. Host cell transcriptional profiling during malaria liver stage infection reveals a coordinated and sequential set of biological events[J].BMC Genomics. 2009.10:270.
32. Arévalo-Rodríguez M, Heitman J. Cyclophilin A is localized to the nucleus and controls meiosis in Saccharomyces cerevisiae[J]. Eukaryotic Cell. 2005.4(1):17-29
33. Bornmann WG, Sirotnak FM, Scheinberg DA.Human mitochondrial peptide deformylase, a new anticancer target of actinonin-based antibiotics[J]. J Clin Invest.2004 114(8):1107-16.
34. Boxem M, Tsai CW, Zhang Y, et al. The C. elegans methionine aminopeptidase 2 analog map-2 is required for germ cellproliferation[J].FEBS Lett. 2004.576(1-2):245-50.
35. Bugli F, Khattab A, Vigneti E, et al. Expression cloning and biochemical characterizations of recombinant Cyclophilin proteins from Schistosoma mansoni[J]. Protein Expression Purification. 1998.12(3):340-6.
36. Capron A, Dessaint JP. Molecular basis of host–parasite relationship: towards the definition ofprotective antigens[J]. Immunol Rev.1989.112:27-48
37. Caffrey CR, Gsell C, Ruppel A. Schistosoma japonicum is less sensitive to cyclosporin A in vivo than Schistosoma mansoni[J].Journal of Parasitology. 1999.85(4):736-9
38. Chai SC, Ye QZ.Metal-mediated inhibition is a viable approach for inhibiting cellular methionine aminopeptidase[J].Bioorg Med Chem Lett .2009.19(24):6862-4
39. Chen L, Rao KV, He YX, Ramaswamy K.Skin-stage schistosomula of Schistosoma mansoni produce an apoptosisinducing factor that can cause apoptosis of T cells[J].J Biol Chem. 2002.277(37):34329-34335
40. Cheng G, Davis RE.An improved and secreted luciferase reporter for schistosomes[J]. Mol Biochem Parasitol .2007.155(2):167-171
41. Cheng GF, Lin JJ, Feng XG, et al. Proteomic analysis of differentially expressed proteins between the male and female worm of Schistosoma japonicum after pairing[J].Proteomics. 2005.5(2):511–521
42. Cheng G, Fu Z, Lin J, et al. In vitro and in vivo evaluation of small interference RNA-mediated gynaecophoral canal protein silencing in Schistosoma japonicum[J].J Gene Med .2009.11(5):412-421
43. Chen X, Xie S, Bhat S, Kumar N, et al. Fumagillin and fumarranol interact with P. falciparum methionine aminopeptidase 2 and inhibit malaria parasite growth in vitro and in vivo[J].Chem Biol .2009.16(2):193-202
44. Chu FH, Chen YR, Lee CH, et al. Molecular characterization and expression analysis of Acmago and AcY14 in Antrodia cinnamomea[J].Mycol Res, 2009.113(5): 577-582.
45. Clare S. Gavigana, Senan P. Kielya, Jocelyne Hirtzlinb, et al. Cyclosporin-binding proteins of Plasmodium falciparum[J].International Journal for Parasitology.2003. 33:987-996
46. Cutforth T, Gaul U.A methionine aminopeptidase and putative regulator of translation initiation is required for cell growth and patterning in Drosophila[J].Mech Dev.1999.82(1-2):23-8
47. Degot S, Le Hir H, Alpy F, et al. Tomasetto C.Association of the breast cancer protein MLN51 with the exon junction complex via its speckle localizer and RNA binding module[J]. J Biol Chem, 2004, 79(32):33702-15.
48. Dewey WC, Ling CC, Meyn RE. Radiation-induced apoptosis: relevance to radiotherapy[J]. Int J Radiat Oncol Biol Phys .1995.33 (4):781-796
49. Ettinger NA, Wilson ME. Macrophage and T-cell gene expression in a model of early infection with the protozoan Leishmania chagasi[J]. PLoS Negl Trop Dis. 2008. 25;2(6):e252.
50. Evans H, Mello LV, Fang Y, et al. Microarray analysis of gender- and parasite-specific gene transcription in Strongyloides ratti[J].Int J Parasitol. 2008.38(11):1329-41.
51. Friedrich N, Santos JM, Liu Y, et al. Soldati-Members of a novel protein family containing microneme adhesive repeat domains act as sialic acid-binding lectins during host cell invasion by apicomplexan parasites[J].J Biol Chem. 2010.285(3):2064-76.
52. Gobert GN, McManus DP, Nawaratna S, et al. Tissue specific profiling of females of Schistosoma japonicum by integrated laser microdissection microscopy and microarray analysis[J].PoS Negl Trop Dis. 2009 .30;3(6):e469.
53. Gobert GN, Moertel L, Brindley PJ, et al. Developmental gene expression profiles of the human pathogen Schistosoma japonicum[J].BMC Genomics. 2009.25;10:128.
54. Gupta BC.Basch PF. The role of Schistosoma mansoni males in feeding and development of female worms[J]. J Parasitology. 1987.73(3):674.
55. Hachet O, Ephrussi A. Drosophila Y14 shuttles to the posterior of the oocyte and is required for oskar mRNA transport[J]. Curr Biol, 2001, 11(21):1666-74.
56. Hachet O, Ephrussi A. Splicing of oskar RNA in the nucleus is coupled to its cytoplasmic localization[J]. Nature, 2004, 428(6986):959-63.
57. Henkle KJ,Cook JA,Foster LA, et al. The gene family encoding eggshell proteins of Schistosoma japonicum[J].Vaccine,1997,15(8):846-848.
58. Horibe T, Yosho C, Okada S, et al. The chaperone activity of protein disulfide isomerase is affected by cyclophilin B and cyclosporin A in vitro[J]. J .Bio chem. 2002;132(3):401-7.
59. Hu X,,Addlagatta A, Lu J, et al. Elucidation of the function of type 1 human methionine aminopeptidase during cell cycle progression[J].Proc Natl Acad Sci U S A 2006.103(48):18148-53
60. Khattab A, Pica-Mattoccia L, Klinkert MQ, et al. Cyclosporins: lack of correlation between antischistosomal properties and inhibition of cyclophilin isomerase activity[J]. Experimental Parasitology. 1998 .90(1):103-9.
61. Kim DM, Ko BS, Ju JW, et al. Gene expression profiling in mouse liver infected with Clonorchis sinensis metacercariae[J].Parasitol Res. 2009 .106(1):269-78
62. Klinkert MQ, Bugli F, Cruz J, et al. Sequence conservation of schistosome cyclophilins[J]. Molecular & Biochemical Parasitology. 1996.30;81(2):239-42
63. Kumar S, Tamura K, Nei M () MEGA3: integrated software for Molecular Evolutionary Genetics. Analysis and sequence alignment[J].Brief Bioinform .2004.5(2):150-163
64. Lee DY, Seto P, Korczak R.DNA microarray-based detection and identification of waterborne protozoan athogens[J]. Microbiol Methods. 2010.80(2):129-33
65. Li BW, Rush AC, Mitreva M, et al. Transcriptomes and pathways associated with infectivity, survival and immunogenicity in Brugia malayi L3[J].BMC Genomics. 2009.15;10:267.
66. Li W, Boswell R, Wood WB. mag-1, a homolog of Drosophila mago nashi, regulates hermaphrodite germ-line sex determination in Caenorhabditis elegans[J]. Dev Biol, 2000, 218(2):172-82.
67. Liu F, Zhou Y, Wang ZQ, et al. The Schistosoma japonicum genome reveals features of host-parasite interplay[J].Nature. 2009.460(7253):345-51.
68. Liu SX, Yongkang H, Guangchen S, et al. Anti-fecundiimmunity to Sohistosoma japonicum induced in Chinese water buffaloes (Bos buffelus) after vaccination with recombinant 26 kDaglutathione -S -transferase(reSjc26GST) [J].Vet Parasitol, 1997.69:39-47.
69. Marfurt J, Smith TA, Hastings IM, et al. Plasmodium falciparum resistance to anti-malarial drugs in Papua New Guinea:evaluation of a community-based approach for the molecular monitoring of resistance[J].Malar J. 2010 .7;9:8.
70. McManus DP,Wong JY,Zhou J, et al. Recombinant paramyosin (rec-Sj-97) tested for immunogenicity and vaccine efficacy against Schistosomajaponicum in mice and water buffaloes[J].Vaccine,2001.20(5-6):870-878.
71. Minning TA, Weatherly DB,et al. The steady-state transcriptome of the four major life-cycle stages of Trypanosoma cruzi[J].BMC Genomics. 2009 .7;10:370.
72. Michael J. Smout, Thewarach Laha,Jason Mulvenna, et al. Granulin-Like Growth Factor Secreted by the Carcinogenic Liver Fluke, Opisthorchis viverrini, Promotes Proliferation of Host Cells[J].PLoS Pathogens,2009,5(10 ) e1000611,1-16
73. Mohr SE, Dillon ST, Boswell RE. The RNA-binding protein Tsunagi interacts with Mago Nashi to establish polarity and localize oskar mRNA during Drosophila oogenesis[J]. Genes Dev, 2001, 15(21):2886-99.
74. 0hta N,Kumagai T,Maruyama H, et al. Research on calpain of Schistosoma japonicum as a vaccine candidate[J].Parasitol lnt.2004.53(2):l75-181.
75. Parma DH, Bennett PE Jr, Boswell RE. Mago Nashi and Tsunagi/Y14, respectively, regulate Drosophila germline stem cell differentiation and oocyte specification[J].Dev Biol, 2007, 308(2):507-19.
76. Park NI, Muench DG. Biochemical and cellular characterization of the plant ortholog of PYM, a protein that interacts with the exon junction complex core proteins Mago and Y14[J].Planta, 2007, 225(3):625-39.
77. Roger Pelle′a, Francis McOdimbaa, Francis Chumaa, et al. The African trypanosome cyclophilin A homologue contains unusual conserved central and N-terminal domains and is developmentally regulated[J].Gene,2002.290:181-191
78. Richter D, Harn DA, Matuschka FR. The irradiated cercariae vaccine model: looking on the bright side of radiation[J].Parasitol Today.1995.11(8):288-293
79. Rycyzyn MA, Reilly SC, O'Malley K, et al. Role of cyclophilin B in prolactin signal transduction and nuclear retrotranslocation[J]. Mol Endocrinol. 2000;4(8):1175-86.
80. Salzet M, Capron A, Stefano GB. Molecular crosstalk in host– parasite relationships: schistosome– and leech–host interactions[J].Parasitol Today.2000.6(12):536-540
81. Sean Dobson,Takiko May,Matthew Berriman, et al. Characterization of protein Ser:Thr phosphatases of the malaria parasite, Plasmodium falciparum: inhibition of the parasitic calcineurin by cyclophilin-cyclosporin complex[J].Molecular and Biochemical Parasitology.1999.167-181
82. Serra EC, Lardans V, Dissous C.Identification of NF-AT-like transcription factor in Schistosoma mansoni: its possible involvement in the antiparasitic action of cyclosporin A[J].Molecular & Biochemical Parasitology. 1999.25;101(1-2):33-41.
83. Sherry, B., Yarlett, N., Strupp, A., et al. Identification of cyclophilin as a proinflammatory secretory product of lipopolysaccharide-activated macrophages[J]. Proc Natl Acad Sci U S A. 1992.89(8):3511-5.
84. Scott JC,McManus DE. Identification of novel 70-kDa heat shock protein-encoding cDNAs from Schistosoma japonicum[J].Int J Parasitol.1999.29(3):437-444.
85. Sugiyama H,Kawanaka M,Kameoka Y, et al. A novel cDNA clone of Schutosoma japonicum encoding the 34,000 Dalton proteins of Schistosoma japonicum[J].Mol Biochem Parasitol,1990.42(1):69-82.
86. Swidzinski JA, Zaplachinski ST, Chuong SD, et al. Molecular characterization and expression analysis of a highly conserved rice mago nashil homolog[J]. Genome, 2001, 44(3):394-400.
87. Salvesen GS, Duckett CS. IAP proteins: blocking the road to death’s door[J].Nat Rev Mol Cell Biol .2002.3(6):401-410
88. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[J].Nucleic Acids Res .1994.22 (22):4673-4680
89. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science .1998.281(5381):1312-1316
90. Upadhya R, Zhang HS, Weiss LM () System for expression of microsporidian methionine amino peptidase type 2(MetAP2) in the yeast Saccharomyces cerevisiae[J].Antimicrob Agents Chemother .2006.50(10):3389-95
91. Van der Weele CM, Tsai CW, Wolniak SM. Mago nashi is essential for spermatogenesis in Marsilea[J]. Mol Biol Cell, 2007.18(10):3711-22.
92. Vicogne J, Cailliau K, Tulasne D, et al. Conservation of epidermal growth factor receptor function in the human parasitic helminth Schistosoma mansoni[J].J Biol Chem.2004. 279(36):37407-37414
93. Veitch NJ, Johnson PC, Trivedi U, et al. Digital gene expression analysis of two life cycle stages of the human-infective parasite, Trypanosoma brucei gambiense reveals differentially expressed clusters of co-regulated genes[J]. BMC Genomics. 2010. 22;11:124.
94. Verity C,McManus DP,Brindley PJ. Vaccine efficacy of recombinant cathepsin D aspartic protease from Sohistosoma japonicum.Parasite lmmunol.2001.23:153-162.
95. Vyacheslav Yurchenko,Zhu Xue,Barbara Sherry, et al. Functional analysis of Leishmania major cyclophilin[J],International Journal for Parasitology 2008.(38):633-639
96. Wang P, Heitman J.The cyclophilins[J].Genome Biol. 2005.6(7):226..
97. Waine GJ,Yang W,Scott JC, et al. DNA-based vaccination using Schistosoma japonicum (Asian blood—fluke)genes eggshell precursor protein[J].Int J Parasitol.1997.27(7):8ll-8l7.
98. Wang L, Utzinger J, Zhou XN. Schistosomiasis control:experiences and lessons from China[J].Lancet .2008.372(9652):1793-1795
99. Wang X, Gobert GN, Feng X,et al. Analysis of early hepatic stage schistosomula geneexpression by subtractive expressed sequence tags library[J].Mol Biochem Parasitol. 2009.166(1):62-9
100.Yang YL, Serrano MG, Sheoran AS, et al. Over-expression and localization of a host protein on the membrane of Cryptosporidium parvum infected epithelial cells[J].Mol Biochem Parasitol. 2009 Nov;168(1):95-101
101.Yoshino TP, Boyle JP, Humphries JE. Receptor-ligand interactions and cellular signalling at the host–parasite interface[J].Parasitology. 2001.123.l:S143-S157
102.Yu F, Li Y, Liu L. Comparative genomics of human-like Schistosoma japonicum genes indicates a putative mechanism for host-parasite relationship[J].Genomics. 2008.91(2):152-157
103.You H, Zhang W, Moertel L, et al. Transcriptional profiles of adult male and female Schistosoma japonicum in response to insulin reveal increased expression of genes involved in growth and development[J].Int J Parasitol. 2009 .39(14):1551-9.
104.Yurchenko, V., Zybarth, G., O’Connor,M., , et al. Active site residues of cyclophilinA are crucial for its signaling activity via CD147 [J].Journal of Biological Chemistry. 2002.277.22959-22965.
105.Yurchenko, V., Constant, S., Bukrinsky, M., Dealing with the family:CD147 interactions with cyclophilins[J].Immunology 2006.117, 301-309.
106.Zeiner GM, Norman KL, Thomson JM,, et al. Toxoplasma gondii infection specifically increases the levels of key host microRNAs[J].PLoS ONE.2010.5(1 )e8742
107.Zhang YY,Taylor MG,Gregoriadis G, et al. Immunogenicity of plaslnid DNA encoding the 62 kDa fragment of Schistosoma iaponicum mysin [J].Vaccine.2000.18(20):2102-2109
108.Zhu C, Wang X, Deinum J, et al. Cyclophilin A participates in the nuclear translocation of apoptosis-inducing factor in neurons after cerebral hypoxia-ischemia[J].Journal of Experimental Medicine. 2007.204(8):1741-45
109.Zhu Y,Ren J,Ham DA, et al. Protective immunity induced with 23 kDa membrane protein DNA vaccine of Schistosoma japonicum Chinese strain in infected C57BL / 6 mice[J].Southeast Asian Trop Med Pub Hehh.2003.34(4):697-701.
110.Zhou Y, Zheng H, Liu F, et al. The Schistosoma japonicum genome reveals features of host–parasite interplay[J].Nature .2009.460(7253):345-351
111.Zeng T, Cai L, Zeng Q, et al. Immunization of mice with cells from juvenile worms of Schistosoma japonicum provides immunoprotection against schistosomiasis[J].Sci China C Life Sci. 2007.50(6):822-30