日本血吸虫蛋白质组学研究进展

随着日本血吸虫(Schistosoma japonicum)基因组学的发展,应用双向电泳、质谱分析及生物信息学分析开展的蛋白质组学研究正成为日本血吸虫研究的前沿领域。生物体中的基因是相对稳定,但是蛋白质的活动随着生命活动的进行表现出动态的变化,并具有对外界环境发生反应的能力。对日本血吸虫进行蛋白质组学方面的研究,能直接地反映其生理功能的过程。这些研究为研制开发抗日本血吸虫病疫苗、新型治疗药物提供了新的途径。论文从日本血吸虫童虫部分差异表达蛋白、成虫蛋白质的性别差异性、药物相关蛋白组学、免疫蛋白组学等方面,对日本血吸虫蛋白质组学研究做了概述。     

血吸虫功能基因组和蛋白质组研究获得重大进展

国家人类基因组南方研究中心、中国疾病预防控制中心寄生虫病预防控制所、上海瑞金医院、复旦大学和美国、澳大利亚的科研人员通力合作,最近在血吸虫功能基因组和蛋白质组研究方面获得重大进展。4月13日,国际权威学术杂志《公共科学图书馆》(Publiclibraryofsciencepathogens)病原卷在网站上发表了相关论文。研究中,科研人员首次采用了系统生物学研究策略和分析方法,利用高通量蛋白质组鉴定技术和策略在世界上第一次针对日本血吸虫不同发育阶段、性别、表皮和卵壳等进行大规模蛋白质鉴定,鉴定血吸虫蛋白质3200多个,这也是我国科学家第一次…     

中国大陆不同地区日本血吸虫雄虫蛋白组分的研究

中国大陆不同地区日本血吸虫雄虫蛋白组分的研究田锷，林邦发，林矫矫，施福恢，朱顺海，石耀军（中国农科院上海家畜寄生虫病研究所）近年来，国内一些实验室对中国大陆一些自然隔离地区的日本血吸虫成虫进行了蛋白质组分分析，发现了一些差别［１，２，３］，这对我国血...     

中华拟德氏吸虫与日本血吸虫的RAPD遗传标志分析

中华拟德氏吸虫(Paradeontacylix sinensis)是冷血吸虫,日本血吸虫(Schistosoma japonicum)是温血吸虫的重要代表。分别对中华拟德氏吸虫和日本血吸虫成虫的全基因组进行随机扩增多态性研究,筛选出31种随机引物,对2种成虫扩增得到189条多态性基因片断,S6、S46对中华拟德氏吸虫全基因组有特异性,分别扩增出一条大小约399bp和695bp的特异性强条带;引物S2对日本血吸虫基因组有特异性,只扩增1条1670bp的特异性强条带。多数引物对中华拟德氏吸虫和日本血吸虫成虫全基因组扩增出大小相似的条带,推测中华拟德氏吸虫与日本血吸虫的亲缘关系密切。     

ELIB诊断黄牛东毕血吸虫病的研究

本研究应用酶联免疫印清技术分析了东毕血吸虫成虫部分纯化抗原中具有免疫活性的蛋白质组分，其中分子量为３５、８０、９２和９４ｋＤ的蛋白质具有较灵敏的反应原性，可做为东毕血吸虫病的诊断抗原。本试验应用此种抗原探索了诊断黄牛东毕血吸虫病的方法，取得了满意结果。     

ELIB诊断黄平东毕血吸虫病的研究

本研究应用酶联免疫印渍技术（ＥＬＩＢ）分析了东毕血吸虫成虫部分纯化抗原中具有免疫活性的蛋白质组分，其中分子量为３５、８０、９２和９４ｋＤ的蛋白质具有较灵敏的反应原性，可做为东毕血吸虫病的诊断抗原。本试验应用此种抗原探索了诊断黄牛东毕血吸虫病的方法，取得了满意结果。     

基于质谱的蛋白质组学技术及其在绒毛用羊中的应用

蛋白质组学(proteomics)是指应用蛋白质分离、鉴定和定量的技术研究蛋白质组的一门学科,与之对应的蛋白质组学技术随之快速发展,基于质谱的蛋白质组学技术更是在相关领域广泛应用。作者主要就蛋白质的相互作用、差异蛋白质组学和蛋白质的定量研究,一些基于质谱的蛋白质组学技术的原理和优缺点,以及绒毛用羊毛囊研究现状和基于质谱的蛋白质组学技术在绒毛用羊中的应用进展等方面进行了综述。最后,对蛋白质组学技术在绒毛用羊毛囊研究中的应用前景做出展望。     

蛋白质组学技术在家鸡中的应用研究进展

蛋白质组学是利用高分辨的蛋白质分离技术和高效的蛋白质鉴定技术,在整体蛋白质水平上动态、定量地研究生命现象及规律的科学,在医学、畜牧学、食品科学及农业研究等领域得到广泛的应用。蛋白质组学方法以大规模、系统化地研究蛋白质特性为目标,旨在解释蛋白质水平上控制复杂生命的分子网络,与其他组学技术相辅相成,成为系统生物学的组成部分。文章介绍了蛋白质组学常用分析策略、质谱检测技术和数据分析方法,在此基础上,综述了蛋白质组学在家鸡重要经济性状差异蛋白鉴定、肉品质、疾病诊断及食品安全检测等领域取得的进展,并总结了多组学联合分析趋势下蛋白质组学技术尚存在的不足。     

日本血吸虫三磷酸腺苷酶(ATPase)的初步研究

本文叙述了日本血吸虫生物膜能量偶联 ATPase,测定了日本血吸虫的ATPase 活性,同时研究了血吸虫各组分中的 ATPase 分布情况。在此基础上,观察了治疗血吸虫药物(酒石酸锑钾)等对血吸虫 ATPase 活力的影响。     

蛋白质组学与神经系统疾病的研究进展

作者阐述了蛋白质组学和蛋白质组学研究方法的最新进展。通过蛋白质组学方法对中枢神经系统的研究有助于阐明CNS疾病发生、发展、转归的网络机制,寻找疾病特异性蛋白质,针对疾病靶点定向合成药物,构建分子药理模型。     

Enhanced protection against the migratory phase,but defective protection against the intestinal phase of Strongyloides venezuelensis infection in cotton rats,Sigmodon hispidus

The protective capacity of the cotton rat, Sigmodon hispidus, against the migratory and intestinal phases of Strongyloides venezuelensis infection was examined. After subcutaneous infection with infective larvae (L(3)), adult worm recovery rates from male and female animals on Day 71 were only 0.10% and 0.06% of initial dose, respectively. To determine whether this enhanced protection was expressed during the migratory phase or the intestinal phase, larval recovery from the lungs of cotton rat was evaluated 3 days after subcutaneous L(3) infection. The larval recovery rate was only 0.5% of initial dose and about 40-fold lower than that from control mice. Protection in the intestine was also evaluated after intraduodenal implantation of adult worms. About 30% of implanted worms became established and worm burden remained constant until Day 28. Despite a high worm burden on Day 28, EPG was about 25-fold lower than the peak count. To evaluate expulsive capacity and monitor the cellular responses in the intestine of cotton rats, adult Nippostrongylus brasiliensis worms were implanted in addition to S. venezuelensis. Cotton rats were unable to expel adult S. venezuelensis worms, even after 21 days of observation. Although the number of mucosal mast cells increased significantly, the intraepithelial migration of mast cells was not observed. In contrast, N. brasiliensis was expelled by Day 6 in association with goblet cell hyperplasia. These results suggest that in cotton rats, the defective intestinal protection against adult S. venezuelensis worms results from dysfunction of mucosal mast cells.     

A comparison of the protein constituents of the major body compartments of the dog roundworm, Toxocara canis

An analysis of the protein profiles of intact worms and isolated tissues of adult male and female Toxocara canis worms was conducted. Soluble proteins recovered from homogenized whole specimens and dissected tissues (body wall, reproductive tract, esophagus and intestine) of T. canis adults from several different canine hosts were separated by size using gradient sodium dodecyl sulfate electrophoresis (SDS-PAGE) and visualized with silver staining. SDS-PAGE profiles of worms from different hosts were found to be virtually identical irrespective of sex or tissue type. Recovered proteins ranged in size from 3.4 to 325 kDa. As expected, variations existed between the protein profiles of different body tissues, with only slight variations between the sexes. The largest number of recovered proteins was present in the female reproductive tract extracts.     

捻转血矛线虫ES24抗原基因的克隆与表达特性分析

根据捻转血矛线虫ES24抗原基因序列(U64793.1)设计1对特异性引物,用RT-PCR方法扩增出大小约为670 bp的DNA片段。将该DNA片段克隆到pMD18-T载体后进行序列测定和分析,结果发现该基因与GenBank中已知的捻转血矛线虫24 ku ES抗原基因的相似性达96%～98%。将该基因的开放阅读框插入pET28a(+)载体中,获得原核表达质粒pET28/ES24,并转化大肠杆菌BL21。重组细菌用IPTG诱导,经SDS-PAGE分析,结果表明该基因获得了表达,重组蛋白分子量大小约为25 ku。用实时荧光定量PCR技术对该基因在捻转血矛线虫的虫卵、第3期幼虫、雌虫和雄虫等不同发育阶段、不同性别虫体内的表达情况进行了定量分析,结果显示ES24基因在雄性成虫中表达量最高,雌虫和虫卵其次,在第3期幼虫中表达最低。     

Setaria digitata in cattle of Thailand identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Adult Thai Setaria worms collected from cattle which were bred, housed and slaughtered in Thailand were morphologically identified as Setaria digitata. Furthermore, in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) adult Thai S. digitata had the same protein profiles as adult Japanese S. digitata, but did not possess the protein with a molecular size of 69 kDa which was confirmed in adult S. marshalli. In addition, there were no differences in the protein profiles between male and female S. digitata. In point of the distribution pattern of the proteins ranging from 73 to 64 kDa revealed by 2D-PAGE, there were no differences between Thai and Japanese S. digitata, and between male and female worms of the species.     

Molecular and functional characterization of a Schistosoma bovis annexin: fibrinolytic and anticoagulant activity

Annexins belong to an evolutionarily conserved multigene family of proteins expressed throughout the animal and plant kingdoms. Although they are soluble cytosolic proteins that lack signal sequences, they have also been detected in extracellular fluids and have been associated with cell surface membranes, where they could be involved in anti-haemostatic and anti-inflammatory functions. Schistosome annexins have been identified on the parasite's tegument surface and excretory/secretory products, but their functions are still unknown. Here we report the cloning, sequencing, in silico analysis, and functional characterization of a Schistosoma bovis annexin. The predicted protein has typical annexin secondary and tertiary structures. Bioassays with the recombinant protein revealed that the protein is biologically active in vitro, showing fibrinolytic and anticoagulant properties. Finally, the expression of the native protein on the tegument surface of S. bovis schistosomula and adult worms is demonstrated, revealing the possibility of exposure to the host's immune system and thus offering a potential vaccine target for the control of schistosomiasis in ruminants.     

Biological and proteomic studies of Schistosoma mansoni with decreased sensitivity to praziquantel

The impact of the laboratory induced Schistosoma mansoni with decreased PZQ sensitivity on the biological performance of its different developmental stages and the concomitant structural changes of adult worms’ total proteins were investigated. PZQ exposed snails showed stoppage of cercarial shedding for eight weeks followed by progressive significant reduction of cercarial production along four successive weeks. In the vertebrate host, in comparison to Schistosoma mansoni susceptible isolate, inoculated cercariae with decreased PZQ sensitivity led to an evident decrease in male to female ratio associated with significant reduction in tissue egg counts and significant increase in dead egg percentage. Significant reduction in the fecundity was also determined. Interestingly, eggs from adult worms with decreased PZQ sensitivity showed two unique features as they found to be smaller and more spherical in addition to the observation of hourglass shaped miracidium in about 10% of the detected mature eggs. Proteomic analysis of adult worms with decreased sensitivity to PZQ using mass spectrometry revealed up-regulation of Ca²⁺ ATPase 2 and Hsp70. This study can point to the increase incidence of the neuroschistosomiasis due to the small size eggs of Schistosoma mansoni with reduced PZQ sensitivity. These worms can also impact the epidemiology in the field. The study can also provide help to elucidate underlying potential molecular mechanisms of resistance that could lead to possible strategies to reverse drug resistance.     

Antigenic differences among the life cycle stages of Oesophagostomum radiatum

Maturation of Oesophagostomum radiatium is characterised by production of different proteins in all the life cycle stages that occur within the bovine host. One-dimensional sodium dodecyl sulphate-polyacrylamide gels stained for total protein, showed that fourth stage larvae (L4) and adult worms have many proteins in common but L4 has a major additional protein (82,000 D); third stage larvae have many different proteins. Analysis of the proteins of each life cycle stage by Western blotting with sera from naturally infected calves showed that each life cycle stage had specific antigenic proteins. The major protein unique to L4 was identified as a developmentally regulated antigen.     

日本血吸虫Wnt受体蛋白SjFz5在不同发育阶段的组织定位及mRNA表达水平分析

为研究日本血吸虫(Sj)Wnt信号分子受体SjFz5(Frizzled5)在Sj生长发育中的作用,本实验对其在不同发育阶段的mRNA转录水平及组织分布进行了检测,并采用定量PCR方法比较SjFz5基因在Sj不同发育阶段间的mRNA水平差异。以7 d童虫cDNA为模板,扩增SjFz5基因编码胞外区CRD(Cystein rich domain)的基因片段,构建pET-SjFz5-CRD表达重组质粒进行原核表达。以纯化的重组蛋白免疫BALB/c小鼠制备多克隆抗体,并进行SjFz5蛋白的组织定位。结果显示:SjFz5基因mRNA在发育早期表达水平相对较高,其中7 d童虫中表达量最高,13 d和18 d童虫下调了约1/3。23 d及其后雌雄虫的表达水平继续下调,雄虫下调比雌虫略显缓慢。整个成虫阶段SjFz5 mRNA维持在一个相对较低的水平。免疫组化结果显示SjFz5蛋白在虫体组织中分布广泛,其中雌雄虫生殖器官组织中的分布最为明显。随着卵巢和睾丸的逐步发育,SjFz5蛋白的量也呈现增加的趋势。SjFz5 mRNA在7 d童虫内表达量最高,SjFz5蛋白在雌雄生殖器官组织中分布最为明显,提示其介导的Wnt信号通路可能参与调节童虫阶段的细胞增殖、器官分化,并可能调节两性生殖细胞的发育。     

Characterization of a novel vaccine candidate and serine proteinase inhibitor from Schistosoma japonicum (Sj serpin)

Serine proteinase inhibitors (serpins) represent an important superfamily of endogenous inhibitors that regulate proteolytic events active in a variety of physiological functions. Immunological screening of a Schistosoma japonicum adult worm cDNA expression library with sera of Microtus fortis, a naturally resistant vertebrate host, has identified one clone that encoded for a sequence homologous to those of the serpin superfamily. The full-length sequence encoding S. japonicum serpin (Sj serpin) was amplified from adult worm cDNA by using 5'-RACE-PCR and subsequently cloned into the prokaryotic expression vector pET28c. The full-length Sj serpin fusion-protein with his-tag was expressed in E. coli, purified by affinity chromatography and used to immunize New Zealand white rabbits. Sj serpin is located on the tegument in S. japonicum adult worms. C57BL/6 mice immunized with Sj serpin induced the production of high levels of specific IgE and IgG1 subclass antibodies as well as a marked IL-4 response. Lymphocyte surface marker analysis revealed proliferation of CD19 expressing B cells, indicating a predominant Th2-type response to Sj serpin. Immunized mice developed moderate protection against infection of S. japonicum as demonstrated by a 36 and 39% reduction in the recovery of adult worms and eggs, respectively. These data suggested a role for Sj serpin as a vaccine candidate or as a novel target for anti-schistosome drugs.     

两种肺吸虫对水貂寄生适应性致病性的研究

目的：比较三平正肺吸虫（Ｅｃ．）与卫氏肺吸虫（Ｐｗ）,在水貂体内发育、分布、寄生适应性及致病性并确定其给主性质。方法：取２种吸虫囊中童虫,经口感染水貂,以任氏液同法分离组织内各型童虫,或直接剖取肺、肝虫囊内虫体,比较２种虫的检出率、各型虫体、寄生部位及致病情况。结果,三平正肺吸虫在貂体发育呈多态性,成虫寄生于肺脏为主,大型童虫在胸腹腔及内脏居我 ,小型童虫则在胸腹肌内,卫氏肺 虫在貂体仅风 童虫,