日本血吸虫SjTOR分子调节补体反应的分子机制及在虫体发育中的作用

Schistosomiasis japonicum, caused by infection with Schistosoma japonicum, is a tropical/ subtropical zoonosis, with a wide distribution in southern china and brings serious dangers. Immune evasion is the adaptation characteristic accumulated in the course of long-term evolution of schistosoma, and is the key to complete the life cycle accompanying the host's immune destruction. Complement reaction is an important defense mechanism of the host in pathogen infection process. Studies have shown that schistosomes can evade the fatal damage of complement cascade of host by some regulatory molecules. CRIT molecule can regulate complement response by inhibitting the formation of C3 convertase. Schistosoma TOR shares similar the conserved C4b binding site with CRIT, so for speculation that schistosoma TOR molecules might have the function of regulating the complement reactions and play an important role during the worm development of the parasites through its strong immune adjustment. In preliminary work，the SjTOR gene has been cloned, and the recombinant rSjTOR-ed1 protein has been expressed and purified, which can be probed by schistosoma-positive mouse serum in western blot test. In this project, real-time quantitative PCR, western blot, immunohistochemistry and other techniques will be to carry out for SjTOR distribution and expression dynamics research , complement hemolysis test , ELISA, ligand binding-blot were intented to analyze the molecular mechanisms of rSjTOR-ed1 that regulate complement cascade of host, RNA interference was employed to investigate its role in the development of parasites in vitro and in vivo. The study results will clearify the molecular mechanisms of SjTOR modulating the host complement cascade response to the infection of schistosoma, and provide the knowledge for further understanding of immune evasion by Schistosoma.

日本血吸虫病是我国重要的人畜共患寄生虫病，危害严重。免疫逃避是血吸虫在长期进化过程中形成的适应性，是其逃避宿主免疫杀伤完成生活史的关键。补体反应是机体防御病原体感染的重要机制。研究表明血吸虫能逃避宿主补体反应的杀伤，具有多个调节补体反应的分子。研究表明CRIT分子通过抑制C3转化酶形成而调节补体反应。血吸虫TOR与CRIT均具有保守的C4b结合位点，推测血吸虫TOR可能具有调节宿主补体反应的功能，并在虫体发育过程中发挥免疫逃避作用。本研究前期已克隆、表达rSjTOR-ed1蛋白，拟利用实时定量PCR、western blot、免疫组化等技术开展SjTOR表达部位和动态变化等研究，应用补体溶血试验、ELISA、配体结合-印迹等分析其调节补体反应的分子机制，利用RNAi研究SjTOR在虫体发育中的作用，研究结果可阐明血吸虫SjTOR调节宿主补体反应的分子机制，为深入理解血吸虫免疫逃避提供知识。

日本血吸虫病是我国重要的人畜共患寄生虫病，危害严重。免疫逃避是血吸虫逃避宿主免疫杀伤完成生活史的关键。补体反应是机体防御病原体感染的重要机制。血吸虫具有调节补体反应分子能逃避宿主补体反应。首次克隆得到SjTOR基因，含四个跨膜结构域，具有保守的C4b结合位点。SjTOR在虫体发育各个时期均有转录和表达，在尾蚴期转录和表达水平最高，在雌虫中的转录明显高于雄虫。SjTOR主要分布于虫体表膜。在虫卵和尾蚴中有表达。构建了重组表达质粒pET-28a-SjTOR-ed1，在大肠杆菌中诱导表达并纯化获得重组蛋白rSjTOR-ed1，分子量约为14kDa。具有良好的免疫抗原性。在两次独立的动物实验中诱导小鼠产生较高水平的特异性IgG抗体，分别获得了24.51%和26.51%的减虫率及39.62%和32.92%的减卵率。设计并合成4对SjTOR基因特异siRNAs，在体外细胞筛选中分别诱导SjTOR基因转录水平降低了70%、63%、3.63%、43%。应用浸泡法对体外培养童虫SjTOR进行干扰，4对siRNAs对TOR基因的转录分别产生了45.12%、38.05%、25.46%、44.25%的干扰效果。将S1、S2、S4siRNA分子通过小鼠尾静脉注射进行体内RNA干扰试验，分别诱导SjTOR基因的转录水平下降84.7%、80.9%和23%。虫荷数和肝脏组织虫卵数分别减少了6.07%和10.06%、24.24%和44.27%、14.02%和63.18%。溶血试验分析比较其各个发育期蛋白对宿主补体活性的影响，结果表明随着虫体的发育，虫体蛋白抑制补体溶血的能力也增强，和SjTOR的相对表达水平是相似的。重组蛋白rSjTOR-ed1能抑制补体溶血且其抑制作用和蛋白量相关，蛋白浓度为10μM时的溶血抑制率可达60.26%。特异性抗血清能在体外试验中显著影响重组蛋白rSjTOR-ed1的溶血抑制功能。补体C2结合试验结果表明，重组蛋白rSjTOR-ed1可与补体C2结合。未观察到rSjTOR-ed1对D因子分解B因子产生Bb亚基有影响。结果表明SjTOR通过与补体C2结合的方式抑制补体活性，该基因虫体发育过程中具有重要作用。研究结果为深入理解血吸虫免疫逃避提供了知识。共发表论文11篇，其中SCI论文8篇，获得发明专利1项，申报发明专利2项，已毕业硕士研究生4名。