环形泰勒虫转化牛B淋巴细胞的转录组及蛋白组学研究

Theileria annulata can transform B lymphocytes and macrophages it infected. Parasitized cells can be immortalized, and acquire the ability to proliferate continuously as cancer cell did. The intracellular protozoan parasite Theileria （T. parva and T. annulata） is the only eukaryote known to induce uncontrolled host cell proliferation. This provide us an intriguing model for the study of parasite-host interactions. This interaction between parasites and host cells is directly in relation to mechanisms underlying pathogenicity and immunity escape(The clinical signs of T. annalata infection is characterized by swelling of the superficial lymph nodes, enlargement of liver and spleen, macrophage proliferation, and lifetime parasite carrier). but the mechanisms underlying this transformation are not well understood.the objectives of the present project are to study the transcriptomic and proteomic profiles of bovine B lymphocytes after infected with T. annulata by an integrated approach using RNA-Seq Solexa high-throughput sequencing, two-dimensional gel electrophoresis, mass spectrometry and bioinformatics analysis combined with fluorescence quantitative PCR, gene cloning and expression, and functional research, to identify differentially expressed genes and proteins in the process of T. annulata sporozoite infection, and to further characterize those representative genes and proteins relating to signal transduction pathway underlying cell immortalization. The expected results would contribute to better understand the T. annulata-host interaction.

环形泰勒虫可以感染牛B淋巴细胞和巨噬细胞，并可使被感染细胞永生化，获得类似癌细胞那样的无限传代能力，泰勒虫是业已发现的、可永生化哺乳动物细胞的、仅有的一类真核生物（如，小泰勒虫和环形泰勒虫）。这种病原与宿主细胞相互作用，与泰勒虫的致病及免疫逃避机理直接相关（环形泰勒虫病表现为体表淋巴结肿胀，巨噬细胞增生与坏死，表现为终生带虫），但到目前为止，对参与这一转化过程的分子及信号转导路径仍不清楚。本项目以环形泰勒虫转化的牛B淋巴细胞为研究对象，通过 RNA-Seq高通量测序、二维电泳、质谱分析、生物信息学分析、荧光定量PCR验证、基因克隆等研究手段，对环形泰勒虫感染牛B淋巴细胞过程中的转录组及蛋白组进行系统研究，鉴定出宿主B淋巴细胞感染环形泰勒虫前后的差异表达基因及蛋白分子，并对其功能进行分析预测，解析宿主细胞实现永生化的Pathway,为系统研究环形泰勒虫与宿主细胞相互作用的机理提供新的线索

构建了环形泰勒虫裂殖体感染细胞的抑制性消减文库，获得大小在350～1200 bp之间的有效表达序列标签（ESTs）364条，与ESTs相匹配的基因序列为192条；完成了环形泰勒虫感染细胞的转录组测序，共获得47.89Gb 数据，开展了生物信息学分析，这些基因按照功能及转录水平存在明显差异分为297类，涉及癌症相关基因、细胞凋亡、B细胞受体细胞通路、p53信号通路、NFKB信号通路等，发现差异表达基因2157个，其中转录水平上调或变化不明显的基因228个、下调基因1928个，对差异表达基因KEGG的注释结果按照KEGG中通路类型进行了分类，发现主要与肿瘤路径、细胞周期调控路径等相关；选取环形泰勒虫的EMM, URP,SUSP2, Cyc, Glu, CP和Tasp等7个基因，通过酵母双杂交技术，获得了与之相互作用的宿主蛋白分子；采用所建立的Real-time PCR检测方法，对3个环形泰勒虫感染细胞的Toll样受体和NOD样受体信号通路上的48个分子的转录水平进行了检测。发表论文8篇；获得甘肃省自然科学三等奖1项；培养博士研究生2名、硕士研究生2名。