II型弓形虫入侵分泌蛋白组学分析和重要分泌蛋白功能鉴定

Toxoplasma gondii，an obligate intracelluar protozoan , can cause serious disease to both human being and animals. Toxoplasma gondii infection puts high risk on human health and food safty, and leads great economic loss to husbandry. Understanding of the invasion and pathogenicity mechanism is crucial for better control of this pathogen. Type II is the major genotype of Toxoplasma gondii to cause human and animal toxoplamosis. Investigation the secretome and fuction of secretory protein during invasion into mice intestinal will contribute to understand its oral-invasion mechanism. Infect mice with oocyst of Type II though oral feeding, and collect the secretory proteins of Toxoplasma gondii in mice small intestine.The secretome of Toxoplasma gondii during invasion will be analyzed by 2D-DIGE and LC-MS/MS. After bioinformatics analysis, a secretory protein which may join into the parasite invasion process will be chosen to study its expression level in different development phases、distribution and localization in vivo and in vitro.Antibody blocking and immune protection experiments will be conducted to investigate whether it influence the Type II Toxoplasma gondii's growth efficiency in vitro ,to know the parasite infection rate and lethality rate to mouse. Immunoprecipitation and LC-MS/MS will be conducted to detect the host proteins interacting with the selected secretory protein. The prospective results of this study will not only be great significance to fundmental research, but also benefit the development of drug and vaccine against Toxoplasma gondii infection.

弓形虫是一种专性细胞内寄生原虫，可引起人畜感染致病，严重危害人类健康和公共食品卫生安全，给畜牧业生产带来巨大经济损失。阐明弓形虫入侵致病机制对弓形虫病的防制至关重要。II型弓形虫是导致人畜弓形虫病的主要基因型之一，开展其在小鼠肠道入侵时的分泌蛋白组和分泌蛋白功能研究有助于解析II型弓形虫经口感染的入侵作用机制。将II型弓形虫卵囊经口接种小鼠，收集其在小鼠小肠入侵时期的分泌蛋白，进行荧光双向差异凝胶电泳分离和质谱检测。经生物信息学分析，选择一个可能与弓形虫入侵相关的分泌蛋白，研究其在弓形虫各个发育期的表达情况及在弓形虫体内外的分布和定位。通过抗体阻断和免疫保护试验，分析其对弓形虫体外生长效率、对感染小鼠致死率荷虫率的影响，通过免疫共沉淀鉴定与该分泌蛋白互作的宿主蛋白，从而阐明该分泌蛋白在弓形虫入侵过程中的作用。预期研究成果不仅具有重要科学价值，而且可为弓形虫药物开发和疫苗研制奠定理论基础。

弓形虫病是世界性流行的重要人兽共患寄生虫病，迄今尚无有效的防控药物和疫苗。弓形虫在入侵和宿主体内生活时，分泌大量蛋白，参与多种重要的生物学过程。阐明这些分泌蛋白的种类、特性和功能，对于了解弓形虫入侵机制、发现新型防控靶分子具有重要意义。本研究首次在小鼠肠道中鉴定了71个在经口感染后6 h时分泌的Ⅱ型弓形虫PRU株蛋白，以及154个在小鼠急性感染期脑组织中转录的弓形虫基因。利用人工合成、经过密码子优化的弓形虫1型蛋白磷酸酶（TgPP1）基因，通过原核表达获得了重组弓形虫1型蛋白磷酸酶（rTgPP1）蛋白，其抗原性良好。发现TgPP1主要定位于弓形虫速殖子顶端；其编码基因在弓形虫感染Hela细胞后5 min时转录水平达最高峰，此后逐渐下降至42 h止，在72 h时又有所上升；在弓形虫由速殖子向缓殖子转化的第1 h转录水平呈现上升趋势，此后逐渐下降至42 h止，在72 h时又上升到较高水平。采用GST pull-down技术筛查与TgPP1互作的小鼠肠道蛋白，共鉴定了281个蛋白，其中丰度较高的为小鼠的磷酸化蛋白。以rTgPP1蛋白为抗原，建立了诊断猪弓形虫感染的间接ELISA法，与商品化试剂盒的阴、阳性符合率在92.0%以上。发现重庆、广州和上海猪膈肌中弓形虫平均阳性率为5.34%。培养毕业研究生3名，发表论文2篇，申请专利1项，参编专著1部。研究结果为深入探究弓形虫TgPP1的特性和功能、研发新型弓形虫病防控技术打下了基础。