旋毛虫丝氨酸蛋白酶介导宿主高度免疫抑制的确证及其分子作用机制

Using cDNA expression library immunological screening technology, we successfully screened highly expressed and stage-specific antigen genes at different developmental stages of Trichinella spiralis (T. spiralis). It was wondering that of the 4 high abundance antigen genes screened in the intestinal phase, 3 genes encoded the serine protease (SP) of different families. But the surprise is that we not only detected this very high SP activity in the excretion and secretion products of adult and newborn larvae in intestinal phase, and found that it has strong abilities of anti-coagulation, inhibition of macrophage proliferation and triggered its apoptosis, more important is all or corresponding abilities were almost completely inhibited or blocked not only by the SP inhibitor phenylmethyl sulfonyl acyl fluoride and anti-T. spiralis serum, but by antibodies against the 3 SP proteins. Given that T. spiralis causes high host immune suppression mainly occurs in the intestinal phase, we speculate that the SP family secreted by adult and newborn larvae has the potential to participate in or mediate this high immunosuppression. .In the project, techniques like in vivo simulation, antibody blocking, worm gene knockdown, animal gene knockout，immune cell and its cytokine transcription, complement degradation and so on will be used to further confirm our hypothetical and explore its molecular mechanism. It is expected to provide new markers, targets or approach for diagnosis, treatment and prevention of trichinosis.

利用cDNA表达文库免疫学筛选技术，我们在旋毛虫不同发育时期均成功地筛选到高表达、且具有期特异性的抗原基因。不解的是，在肠道期筛选到的4个高丰度抗原基因中，有3个基因均编码不同家族的丝氨酸蛋白酶（SP）。让人惊喜的是，我们在肠道期成虫与新生幼虫的排泄分泌物中均检测到了这种非常高的SP活性，且发现其具有很强的抗凝血、抑制巨噬细胞增殖并引发其凋亡的能力，尤为重要的是这些能力不但可被SP抑制剂苯甲基磺酰氟，更可被旋毛虫感染血清及抗这3个SP蛋白的抗体全部或相应的抑制或封闭。鉴于宿主的高度免疫抑制主要发生于旋毛虫的肠道期，我们推测成虫与新生幼虫分泌的SP具有参与或介导这一高度免疫抑制的可能。本课题拟通过体内模拟，抗体阻断，虫体基因敲减及动物基因敲除，免疫细胞及其细胞因子转录，补体切割等技术来进一步确证这一假想并探究其可能的分子机制。可望为旋毛虫病的诊断、治疗及预防发现新标识、靶点及途径。

研究表明，肠道期旋毛虫可对宿主免疫进行调节，使宿主免疫产生较强的抑炎反应，不仅可确保虫体尽可能地避免来自宿主炎性免疫反应的攻击，同时也同时也最大限度地减少了因虫体侵袭引发的宿主炎性反应对宿主本身的损害（避免炎性风暴的发生）。团队在前期研究中筛选鉴定了3个不同家族的丝氨酸蛋白酶，且发现其具有很强的抗凝血、抑制巨噬细胞增殖并引发其凋亡的能力。.本研究在前期研究基础上，制备了三种丝氨酸蛋白酶单克隆及多克隆抗体，证实相关酶活性及催化底物特异性。阐明了旋毛虫丝氨酸蛋白酶TspE1对宿主免疫细胞的调控机制，TspE1通过诱导未成熟树突状细胞（BMDCs）表面MHCII和CD86分子的表达上调，促进BMDCs的成熟，通过NLRP3信号通路，使IL-1β、IL-18,、IL-10和TGF-β分泌量显著升高；诱导后的DC与初始CD4+ T细胞共孵育后，可使初始CD4+ T细胞向Th2和Treg方向分化。TNBS诱导的小鼠结肠炎的动物模型中，旋毛虫TspE1诱导的DC可以有效缓解小鼠肠道炎症，并且模型小鼠体内的CD4+ T细胞由Th1型转变为Th2型和Treg主导的免疫应答。相关研究为旋毛虫病的诊断、治疗及预防发现新标识、靶点及途径。.