鸡柔嫩和巨型艾美尔球虫种特异性保护性抗原的筛选鉴定及其免疫特异性分子机制的研究

Coccidiosis is the most economically important parasitic disease of poultry industry. Rapid development of drug-resistant strains of Eimeria species and drawbacks of high costs and environmental contaminations of the live vaccines against Eimeria species make more challenges the prevention and control of coccidiosis. To develop the polyvalent subunit based third-generation vaccine that is expected to be safer, lower costs, and more efficacy, systematic screening and identification of the species-specific protective antigens of chicken coccidia, as well as elucidating the molecular mechanisms of the specific immune responses induced by these antigens is the most critical step. In the proposed research project, the E. tenella, a species with the strongest pathogenicity and a weak antigenicity, and the E. maxima, a species with the strongest antigenicity and a moderate pathogenicity are chosen as experimental species, using immuno-proteomics analysis combining with transcriptomics, comparative proteomics, bioinformatics, genetic engineering technologies, and animal immune protective experiments, systematic screening and identifying species-specific protective antigens from different developmental stages of the two chicken Eimeria species; to analyze and compare comprehensively the two different groups of the coccidia protective antigens; to study molecular mechanisms of host specific immunity induced by species-specific antigens at levels of genes/proteins, functional domains, and nucleotides/amino acids; to reveal mechanisms of host specific immune responses and their relationship with anti-coccidia infections. The research methodologies, procedures and models for related researches will be established to provide a theoretical basis and technical support for the development of novel multivalent and efficient vaccines against chicken coccidia.

鸡球虫病是危害养鸡业最严重的寄生虫病，耐药虫株的出现和活虫疫苗生产成本昂贵及污染环境的缺点，使鸡球虫病的防控面临更严峻的挑战。为研发安全低廉、多价高效的第三代分子疫苗打下基础，系统筛选鉴定鸡球虫种特异性保护性抗原，阐明其诱发特异性免疫的分子机制，是最关键的一步。本项目拟选用致病性最强、抗原性较弱的鸡柔嫩和抗原性最强、致病性中等的巨型艾美尔球虫为代表，应用免疫蛋白质组学技术，结合转录组学、比较蛋白质组学、生物信息学技术、基因工程技术及动物免疫保护试验，从两种鸡球虫的不同发育阶段虫体中系统的筛选鉴定该两种球虫的种特异性保护性抗原；综合分析比较两种球虫的保护性抗原的差异； 从基因/蛋白质、功能区、碱基/氨基酸水平上研究种特异性抗原诱发宿主种特异性免疫的分子机制；揭示宿主特异性免疫应答机制及其与抗感染的关系。建立相关的研究方法、程序和模型，为研发多价高效新型鸡球虫疫苗提供理论依据和技术支持。

项目背景：孢子化鸡球虫卵囊感染可以诱导鸡体建立完全免疫保护；不同种鸡球虫交叉免疫保护较弱。这提示不同种鸡球虫存在种特异性保护性抗原。本研究以柔嫩和巨型艾美尔球虫为研究对象，应用比较免疫蛋白质组学对两种鸡球虫种特异性抗原进行筛选鉴定；通过鉴定种特异性抗原关键序列、与宿主的互作关系阐述其参与抗感染的机制。.研究内容：1. 鸡柔嫩和巨型艾美尔球虫种特异性抗原的筛选；2. 鸡柔嫩艾美耳球虫和巨型艾美尔球虫种特异性抗原鉴定；3. 鸡柔嫩和巨型艾美尔球虫特异性抗原关键功能区鉴定；4. 鸡艾美尔球虫特异性免疫的分子机制研究。.重要结果、关键数据及其科学意义：. 1、筛选了柔嫩艾美尔球虫种特异性抗原组：成功制备了柔嫩和巨型艾美尔球虫高免血清；应用比较差异免疫蛋白质组学技术，筛选了109个免疫差异蛋白点。2、克隆、表达了柔嫩艾美尔球虫差异抗原：表达了7个差异基因，制备了多克隆抗体，鉴定了3个新的子孢子表面抗原（EtCCT2、EtHSP60、EtPyruvate kinase）。3、鉴定了3个鸡柔嫩艾美尔球虫和3个巨型艾美尔球虫种特异性抗原：通过体内体外免疫原性及交叉免疫保护力的综合评价试验，鉴定了鸡柔嫩和巨型艾美尔球虫种特异性抗原各3个：EtCCT2、EtMIC1、EtProfilin；EmHSP60、EmRACK、EmSERPIN1。同时鉴定了1个共同抗原（Pyruvate kinase）。4、鉴定了差异抗原（EtMIC1/EtMIC2）的关键表位/结构域：鉴定了EtMIC1的一个关键表位（91LITFATRSK99）；发现I-domain和TSP2结构域是EtMIC1诱导保护性免疫反应的关键结构域；鉴定了EtMIC2的一个抗原表位：241KEGMCEADDPELIALTR257。5、鉴定了柔嫩和巨型艾美尔球虫CCT2的功能差异：对EtCCT2/EmCCT2与宿主的互作关系进行了分析，结果发现，EtCCT2与鸡GgCCT4和GgCCT5互作，互作复合物以颗粒状的形式聚集、入核，最终导致核裂解，细胞凋亡增加；而EmCCT2不与鸡GgCCT4互作。本研究应用免疫差异蛋白质组学对柔嫩艾美尔球虫种特异性抗原进行筛选鉴定，结合动物实验，鉴定了鸡柔嫩和巨型艾美尔球虫种特异性抗原各3个；并对特异性抗原关键表位及与宿主的相互作用机制进行解析，为柔嫩艾美尔球虫新型亚单位疫苗的研发提供了理论依据。