【作者】 张旻；

【导师】 林矫矫；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2014， 博士

【摘要】 日本血吸虫病是一种分布广泛、危害严重的人畜共患寄生虫病。体被是血吸虫和宿主物质交换的场所，也是宿主免疫效应分子与虫体直接接触的界面，与血吸虫营养摄取、信号传导和免疫逃避等密切相关。一些体被表膜蛋白，特别是期别和性别差异表达体被表膜蛋白对维持血吸虫在终末宿主体内寄生及完成其生活史至关重要。开展日本血吸虫体被表膜蛋白质的组成和变化分析，及体被蛋白质组的免疫学筛选，鉴定与日本血吸虫生长发育相关的重要分子，及免疫相关分子，可为阐述血吸虫与宿主的相互作用机制提供重要信息，为疫苗候选分子、新治疗药物靶标和诊断抗原分子的筛选提供新思路，具有重要的理论价值和潜在的应用前景。1．日本血吸虫童虫与成虫体被表膜蛋白质组分析血吸虫的生活史复杂，在不同发育阶段表现出不同的生物学和形态学特征。童虫和成虫体被表膜上差异表达的蛋白，对血吸虫的存活、生长和繁殖具有重要意义。本研究应用非渗透性生物素sulfo-NHS-SS-biotin标记14d童虫和32d成虫的体被表膜蛋白，利用链酶亲和素-生物素系统纯化获得生物素标记蛋白，随后利用液相色谱串联质谱法进行高通量蛋白质组学分析。结果显示，童虫和成虫分别鉴定到了245个和103个蛋白分子，包括59个共有的蛋白，186个童虫高表达的蛋白，及44个成虫高表达的蛋白。90%的差异表达体被表膜蛋白的理论相对分子量为10-70kDa，等电点为4-10。GO分析显示，童虫高表达蛋白主要与RNA代谢过程，基因表达，大分子生物合成过程，RNA结合等相关。而成虫高表达蛋白主要与中性粒细胞凋亡过程的调节，过氧化氢分解代谢过程，丝氨酸型肽酶活性等相关。本研究为揭示血吸虫在终末宿主体内的生长发育机制提供重要信息，为筛选血吸虫疫苗候选分子、药物靶标以及诊断抗原分子提供新思路。2．日本血吸虫雌雄虫体被表膜蛋白质组分析雌雄虫之间的相互作用是雌虫发育成熟以及产卵的先决条件，也与血吸虫的致病和传播有着紧密联系。一些雌雄虫差异表达蛋白在虫体与宿主的相互作用中具有重要的作用。本研究利用链霉菌亲和素-生物素亲和纯化技术以及液相色谱-质谱/质谱联用技术分析了日本血吸虫雌、雄虫体被表膜蛋白质组，二者分别鉴定到了179个和300个蛋白分子，包括119个雌雄虫共有的，60个雌虫高表达的，和181个雄虫高表达的蛋白。生物信息学分析表明，在这些被鉴定的体被表膜蛋白中，有的参与到了血吸虫与宿主的相互作用中，如serpin和CD36-like class B scavenger receptor，有的与雌雄虫之间的相互作用有关，如gynecophoral canal protein。GO分析显示，雌虫高表达蛋白主要与蛋白质糖基化和溶酶体功能等相关，而雄虫高表达蛋白主要与细胞内信号转导、丝状肌动蛋白聚合作用的调控、蛋白酶体复合物功能等相关。该研究结果为分析明确雌雄虫在生理学上的差异及更好地理解雌雄虫相互作用和性成熟机制提供重要的实验依据，同时也可为抗血吸虫病疫苗候选分子和药物靶标的筛选提供基础。3．基于体被抗原免疫蛋白质组学的日本血吸虫病诊断抗原的筛选及应用价值评估由于体被直接暴露于宿主内环境，一些体被抗原分子成为刺激、诱导宿主产生特异性免疫应答的重要分子。本研究以日本吸虫体被蛋白作为抗原，以日本血吸虫感染前，感染后的2周和6周，以及吡喹酮治疗后1，2，3，4，5，6，7，8月的兔血清为一抗，利用免疫蛋白质组学技术筛选具有免疫诊断价值的抗原分子。研究结果显示，有10个蛋白点未被感染前兔血清识别，但在感染后的第2周和6周都呈阳性反应，而在治疗后的早期阶段又转为阴性反应。经质谱鉴定，这10个蛋白点分属于6个不同蛋白质。我们从中挑选磷酸甘油酸酯变位酶（Phosphoglyceratemutase，PGM）进行了克隆、表达。经免疫组织定位分析表明SjPGM为一体被蛋白。ELISA法分析结果显示，实验兔感染日本血吸虫2周后，所有兔子都可检测出抗rSjPGM和SEA的特异性抗体。在吡喹酮治疗后的第2月至第7月，以rSjPGM作为诊断抗原，所有实验兔都陆续转为阴性，而直至吡喹酮治疗后第8月，以SjSEA作为诊断抗原，所有实验兔仍都呈阳性。表明在考核药物疗效，或区分现症感染和既往感染方面，rSjPGM作为诊断抗原要明显优于目前最常用的SjSEA。进一步应用ELISA法检测了104份血吸虫感染阳性水牛血清和60份健康水牛血清，结果以rSjPGM和SjSEA作为诊断抗原，其敏感性分别为91.35%和100.00%，特异性分别为100.00%和91.67%。而在检测14份前后盘吸虫、9份大片吸虫感染水牛血清时，rSjPGM的交叉反应率为7.14%和11.11%，SjSEA为50.00%和44.44%，表明rSjPGM作为牛血吸虫病的诊断抗原具有潜在的应用价值。4．日本血吸虫SjCHMP5的克隆、表达及rSjCHMP5诱导抗血吸虫感染免疫保护效果的评价在前期体被蛋白质组学研究的基础上，挑选出一体被蛋白带电多泡体蛋白5（Chargedmultivesicular body protein5，CHMP5）进行克隆、表达、生物学特性分析及重组蛋白诱导抗血吸虫感染免疫保护效果的评价。利用PCR技术从日本血吸虫成虫cDNA文库中克隆到了编码SjCHMP5（GenBank accessionno. FN320038）的ORF序列，该序列含675bp，编码224个氨基酸，其理论等电点和分子量分别为4.49及25kDa。构建了该基因的重组表达质粒pET28a(+)-SjCHMP5，在大肠杆菌系统中成功获得表达，重组蛋白rSjCHMP5以可溶性蛋白形式存在，其理论分子量约为29kDa。质谱鉴定结果表明，检测的纯化重组蛋白确实为rSjCHMP5。Western blotting分析表明，rSjCHMP5具有较好的抗原性。免疫荧光染色试验分析显示，SjCHMP5主要定位于日本血吸虫虫体的体被上，为一体被蛋白。应用纯化的rSjCHMP5结合206佐剂免疫BALB/c小鼠，与佐剂对照组相比，诱导了18.96%的减虫率（P<0.05）和42.98%的肝脏减卵率（P<0.01）。ELISA试验表明，重组蛋白二次免疫后针对rSjCHMP5的特异性IgG抗体滴度即达到较高水平。本研究表明rSjCHMP5可诱导小鼠产生部分的抗血吸虫病的免疫保护效果。综上，本研究首次应用生物素链酶亲和素系统分离纯化了日本血吸虫童虫、成虫以及雌雄虫体被表膜蛋白，应用Shotgun技术和生物信息学技术系统分析其组成和变化。同时，首次应用免疫蛋白组学技术，比较分析血吸虫感染前、感染后以及经吡喹酮治疗后不同时期具有免疫原性的血吸虫成虫体被抗原的变化情况。筛选出一个具有血吸虫病诊断价值的抗原分子SjPGM。首次对日本血吸虫体被蛋白SjCHMP5编码基因进行了克隆、表达及生物学特性分析，评估了其重组蛋白在小鼠体内诱导的抗血吸虫感染的免疫保护效果。研究成果为鉴定血吸虫与宿主相互作用的关键分子、阐述日本血吸虫的生长发育机制提供了有价值的信息，为抗血吸虫病疫苗候选分子、新药物靶标和新诊断抗原分子的筛选提供了新思路，具有重要的理论价值和潜在的应用前景。更多还原

【Abstract】 Schistosomiasis is a kind of widely distributed and seriously harmful zoonotic parasitic diseases.Schistosome tegument is not only the place for exchanging materials with host but also the interface indirect contact with the the host immune effector molecules, and closely associated with nutritionalintake, signal transduction and immune evasion. Some tegument-exposed proteins, especiallydifferential expression between stages or genders, should be important for sustaining parasitism andcompleting its lifecycle in the definitive hosts. Proteomic analysis on the composition and alteration oftegument-exposed proteins of Schistosoma japonicum, immunoproteomics study on the tegumentproteins, and identification of important molecules associated with the growth, development andimmunity of worms, will not only provide important information for elaboraing mechanisms aboutinteraction between host and schistosome, but also provide new ideas for screening vaccine candidates,new drug targets and diagnostic antigens. This study has important theoretical values and potentialapplications.1. Proteomic analysis of tegument-exposed proteins of schistosomula and adult Schistosomajaponicum wormsSchistosome has a complex lifecycle and exhibits dramatic changes in its biology and morphologyat different developmental stages. Differentially expressed tegument-exposed proteins betweenschistosomulum and adult worm should be important for survival, development, and reproduction of theparasites. In this present report, we conducted an incubation of intact and live14-d-old schistosomulaand32-d-old adult with a membrane-impermeant sulfo-NHS-SS-biotin to label tegument surfaceexposed proteins and later recovered the biotinylated proteins using streptavidin affinity beads, whichwere then subjected to high-throughput proteomics analyses, involving liquid chromatography andtandem mass spectrometry. In total,245proteins were identified in schistosomulum and103in adult,including59proteins common to both stages, and186schistosomulum biased and44adult biasedproteins. For both samples, approximately90%proteins were distributing in a range of MW10-70kDaand pI4-10. Gene Ontology analysis revealed that proteins involved in RNA metabolic process, geneexpression, macromolecule biosynthetic process, RNA binding and other GO terms were highlyexpressed in schistosomulum. In addition, proteins involved in regulation of neutrophil apoptoticprocess, hydrogen peroxide catabolic process, serine-type peptidase activity and other GO terms werehighly expressed in adult. Our study provide new insights into schistosome development and growth inthe final host and permit the screening of novel vaccine candidates or drug targets and diagnosticantigens for schistosomiasis.2. Proteomic analysis of tegument-exposed proteins of female and male Schistosoma japonicumwormsThe interplay between sexes is a prerequisite for female growth, reproductive maturation and eggproduction, and the basis of schistosome pathopoiesis and propagation. Differentially expressed tegument-exposed proteins between female and male worms should be important for the interactionbetween host and schistosome. In this study, a streptavidin-biotin affinity purification techniquecombined with LC-MS/MS was used to analyze putative tegument-exposed proteins in female and maleadult Schistosoma japonicum worms. In total,179proteins were identified in females and300in males,including119proteins common to both sexes, and60female biased and181male biased proteins. Some(e.g., serpin and CD36-like class B scavenger receptor) were involved in host-schistosome interactions,while some (e.g., gynecophoral canal protein) were important in the interplay between sexes. GeneOntology analysis revealed that proteins involved in protein glycosylation and lysosome were highlyexpressed in females, while proteins involved in intracellular signal transduction, regulation of actinfilament polymerization and proteasome core complex were highly expressed in males. These resultsmight elucidate physiological differences between sexes. Our study provide new insights into theinterplay between sexes and sexual maturity in the final host and permit the screening of vaccinecandidates or drug targets for schistosomiasis.3. Screening the diagnostic candidates from tegument proteins of adult Schistosoma japonicum byimmunoproteomics approach and evaluating their applied valueSince the tegument (TG) is in direct contact with the host environment, some tegument proteinscould stimulate and induce specific immunity response in host. In this study, an immunoproteomicsapproach, using TG proteins as antigens and sera collected from rabbits at stages of uninfected, infectedwith S. japonicum and post-treatment of praziquantel as the primary antibodies, was applied to identifythe potential diagnostic molecules of S. japonicum. In total, ten spots corresponding to six differentproteins could not be recognized by uninfected sera but could react with sera from rabbits2and6weekspost S. japonicum infection, and could not be detected again soon after the praziquantel treatment.Among them, SjPGM, identified as a tegument protein by immunofluorescence, was expressed and itsrecombinant proteins was compared to the soluble egg antigen (SEA) by ELISA. In our results, bothspecific IgG antibody against rSjPGM and SjSEA were detected up to positive cut-off values throughoutthe infection process in all rabbits. As for rSjPGM, its sera IgG titer in all ones wereschistosomiasis-negative at2-7months post-treatment. Meanwhile, the level of anti-SEA IgG antibodyremained in positive ranges until8months post-treatment. Moreover, when rSjPGM and SjSEA wereused to examine sera of104schistosomiasis and60non-infected buffaloes, the results revealed that thesensitivity of them were91.35%and100.00%, and the specificity of them were100.00%and91.67%.In addition, these two kind of antigens were also detected in the testing serum from Paramphistomum orFasciola gigantica–exposed individuals, the crossing reactivity in them with a proportion of7.14%and11.11%for rSjPGM and50.00%and44.44%for SEA. Our study reveal that rSjPGM have potentialapplications as diagnostic antigen for buffaloes schistosomiasis.4. Cloning, expression and immunoprotection of CHMP5in Schistosoma japonicumBased on the previous tegument proteomics research, one protein charged multivesicular bodyprotein5(CHMP5) were selected for further study.In this study, the cDNA encoding the Schistosoma japonicum CHMP5(SjCHMP5) was amplified by PCR. The cDNA had an open reading frame (ORF) of675bp and encoded224amino acids. TheSjCHMP5was successfully subcloned into pET28a(+) and pET28a(+)-SjCHMP5was expressed assoluble proteins of29kDa in E. coli BL21(DE3) cells. The result of mass spectrum revealed that thepurified proteins was indeed rSjCHMP5. Western blotting showed that rSjCHMP5had good antigenicity.Immunofluorescence analysis revealed that SjCHMP5mainly distributed on the tegument of worms andwas a tegument protein. Compared with the adjuvant group, BALB/c mice immunized with rSjCHMP5combined with Seppic206adjuvant three times showed a18.96%worm reduction (P<0.05) and a42.98%egg count reduction (P<0.01). ELISA tests showed that specific IgG antibody titers ofrSjCHMP5reached a high level after the second immunization. This study suggested that rSjCHMP5provide partial immunoprotection against schistosomiasis.In summary, a streptavidin-biotin affinity purification technique combined with shotgun was usedto analyze putative S. japonicum tegument-exposed proteins in schistosomulum, mixed adult worm,female and male adult worms. In additon, an immunoproteomics approach, using TG proteins asantigens and sera collected from rabbits at stages of uninfected, infected with S. japonicum andpost-treatment of praziquantel as the primary antibodies, was applied to analyze alteration ofimmunogenic molecules and obtain a potential diagnostic molecules, SjPGM, for schistosomiasis. Inaddition, SjCHMP5, identified as a tegument protein, was firstly cloned and expressed. Immuneprotection provided by rSjCHMP5was assessed in mouse model. Our study will not only provide newinsights into the the discovery of key factors in schistosome-host interactions and in-depth knowledge ofschistosome development and growth mechanism, but also permit the screening of novel vaccinecandidates or drug targets and diagnostic antigens for schistosomiasis. This study has importanttheoretical values and potential applications.更多还原