羊泰勒虫种内（间）差异表达蛋白的鉴定和功能预测

Ovine and caprine theileriosis is caused by genus Theileria, which is transmitted by ticks and parasitized in macrophages, lymphblasts and eryhrocytes of sheep and goats. Theileriosis is widespread and seriously affects animal health, and causes a huge economic loss to the global small ruminant industry. Up to date, the disease has not been effectively prevented and controlled, and the main reason is that the basic research is insufficient, especially for all kinds of the differential proteins of proteome about Theileria spp., and their relationship between pathogenesis and the virulence is not clear. In the project, T. luwenshuni and T. uilenbergi, which are infective to sheep and goats, will be selected as the research object, and the comparative studies about proteome of Theileria spp. will be carried out. Through two-dimensional electrophoresis, mass spectrometry analysis, bioinformatics analysis, and gene cloning and expression, function research technology, the differential proteins of proteome among T. luwenshuni strains from different place, and that of T. uilenbergi strains from different place, and that between T. luwenshuni and T. uilenbergi will be identified, respectively, and their functions will be predicted, and their immunogenicity and reactogenicity about differential proteins will be clarified. Research results maybe find new "target molecules" for the immune prevention or chemical prevention and control about ovine and caprine theileriosis. The project research has important academic value and the potential use of the future.

羊泰勒虫病是由媒介蜱传播的由泰勒科泰勒属的原虫寄生于绵羊和山羊巨噬细胞、淋巴细胞和红细胞内所引起的疾病的总称。本病分布广泛、危害严重，给全球养羊业造成巨大经济损失。目前，羊泰勒虫病尚未得到有效防治的主要原因是对羊泰勒虫病防控的基础研究不足，特别是对各种羊泰勒虫在蛋白质组的差异以及其与致病和毒力的关系尚不清楚。本项目选用对绵羊和山羊危害十分严重的吕氏泰勒虫和尤氏泰勒虫为研究对象，开展吕氏泰勒虫和尤氏泰勒虫蛋白质组的比较研究，通过二维电泳、质谱分析、生物信息学分析、以及基因克隆、表达、功能研究等技术手段，鉴定出在吕氏泰勒虫不同地方株之间、尤氏泰勒虫不同地方株之间、吕氏泰勒虫和尤氏泰勒虫虫种之间差异表达的蛋白质、预测其功能，并阐明代表性毒力相关差异表达蛋白的免疫原性和反应原性。研究成果可能为羊泰勒虫病的免疫预防或化学防治找到新的"靶分子"。因此本项目研究具有重要学术意义，已具有潜在的应用前景。

羊泰勒虫病是由媒介蜱传播的由泰勒科泰勒属的原虫寄生于绵羊和山羊巨噬细胞、淋巴细胞和红细胞内所引起的疾病的总称。本病呈全球分布、危害严重。目前，该病尚未得到有效防治的主要原因是对羊泰勒虫病防控的基础研究不足，特别是对各种羊泰勒虫在蛋白质组的差异及其致病和毒力的关系尚不清楚。本研究选取2个种6株不同地方株致病力较强的羊泰勒虫种株作为研究对象进行蛋白质组学分析，分别提交并公布了吕氏泰勒虫及尤氏泰勒虫的全蛋白质组学数据库。通过iTRAQ技术测定了吕氏泰勒虫及尤氏泰勒虫种内及种间的差异表达蛋白，对一些重要功能性蛋白进行了初步分析及转录水平的验证，从吕氏泰勒虫裂殖子克隆TlSP基因，表达和纯化了重组rTlSP抗原。经过真核转染用激光共聚焦显微技术对其在Vero细胞中表达蛋白进行定位。建立了检测羊泰勒虫的间接ELISA方法。TlSP蛋白是寄生虫侵袭宿主细胞的主要蛋白，以TlSP蛋白为诱饵蛋白，利用酵母双杂交技术从羊淋巴细胞文库中调出与其相互作用的蛋白，通过研究相互作用蛋白的功能，探究出吕氏泰勒虫TlSP蛋白与虫体入侵宿主细胞的关系。本研究利用多重PCR和间接ELISA，首次证实吕氏泰勒虫和尤氏泰勒虫除感染绵羊和山羊外，也可以感染黄羊，狍（犬鹿）、马鹿、梅花鹿、黄牛、牦牛和犏牛等多种动物。吕氏泰勒虫和尤氏泰勒虫感染小鼠实验表明，尽管这两种泰勒虫可以感染昆明小鼠和BALB/C小鼠，然而感染小鼠的虫血症很低0.01%-0.03%，感染小鼠无明显病理变化（如无贫血和黄疸等变化），所以小鼠不宜作为研究羊泰勒虫的实验动物模型。但是小鼠作为保虫宿主，在自然界泰勒虫的传播中具有非常重要的科学意义。运用生物信息学初步分析了羊泰勒虫蛋白组的核酸合成代谢路径，进行了乳清酸磷酸核糖转移酶（OPRT）基因的克隆表达，以及 rOPRT 的绵羊保护性效果评价。组建TlSP重组亚单位疫苗，评价了该疫苗对羊泰勒虫的动物保护效果评估试验。本研究提交的数据和结果为今后羊泰勒虫的组学、入侵机理、疫苗免疫等研究奠定了坚实的基础。