旋毛虫移行期幼虫表面结合宿主免疫球蛋白在免疫逃避中的作用

Trichinelle spiralis is a serious parasitic zoonotic parasite. Currently, it is unclear that weather some larval surface proteins mediated the acquisition of host IgG/IgM via the interaction with the Fc domain. In this project, we will detect the binding of non-specifcal immune IgG and IgM on the surface of T. spiralis migratory larvae by IFT, then the surface proteins binding to IgG and IgM were subsequently identified by mass spectrometry. The surface protein which may participate the process of immune evasion will be molecularly cloned and expressed in vitro, and the expression phase and tissue location of these surface proteins will be investigated by qPCR, Western blot and confocal microscopy. By using ELISA and pull down, the specificity of protiens binding to Fab/Fc domain of the immunoglobulin will be identified, and the binding of specific IgG to recombinant proteins which will be blocked or not by the Fc domain of the non-specific immune IgG/IgM will be verified. By migratory larval killing and animal experiment, the function of these recombinant proteins binding to Ig Fc domain in immunocomplex will be identified by the vitro model of T. spiralis ADCC. This project will have an important sentifical significance for illustrating the immune evasion mechanism mediated by larval surface proteins binding host IgG/IgM through avoiding host immune regcognition and attachment. It also provides the basis for the development of vaccine against trichinellosis .

旋毛虫是一种重要的人兽共患寄生虫，虫体表面是否存在能与宿主IgG/IgM Fc段结合的蛋白，参与虫体逃避宿主的免疫识别与杀伤目前尚不清楚。本项目将收集旋毛虫移行期幼虫，应用免疫荧光试验鉴定虫体表面与宿主IgG、IgM的结合，将与IgG、IgM结合的表面蛋白进行质谱分析，挑选具有潜在免疫逃避功能的虫体表面蛋白进行克隆与体外表达，应用qPCR、Western blot及共聚焦显微镜等鉴定其表达时相及组织定位。应用ELISA、pull down等鉴定重组蛋白与宿主Ig的结合能力及片段，分析重组蛋白结合Ig Fc段后对抗重组蛋白抗体结合的阻断或抑制作用，通过幼虫体外杀伤实验及动物实验，观察重组蛋白与免疫复合物中Ig Fc段结合后对ADCC杀虫效果的阻断或抑制作用，评价虫体表面蛋白在旋毛虫免疫逃避中的功能。该项目对于阐明旋毛虫免疫逃避的机制具有重要的科学意义，并可为研制抗旋毛虫疫苗等奠定基础。

旋毛虫病是一种危害严重的人兽共患寄生虫病，其致病主要是幼虫在移行过程中引起人体全身中毒症状及超敏反应。目前，旋毛虫在宿主体内免疫逃避机制仍然不清楚。本项目通过对旋毛虫蛋白质谱鉴定，挑选出具有潜在免疫逃避功能的组织蛋白酶L（TsCL）进行研究，TsCL属于半胱氨酸蛋白酶，分子对接显示其与E64之间存在疏水作用和氢键。体外克隆并表达获得重组TsCL（rTsCL）, qPCR、免疫印迹和荧光结果显示，TsCL在旋毛虫各虫期均有转录与表达，主要定位于虫体表皮、杆状体、肠道和胚胎。应用底物Z-Phe-Arg-AMC检测rTsCL酶活性，结果显示最适温度为55℃、pH为4.5，最大反应速率为573nM/min，米氏常数为62μM/L；Mn2+、Cu2+、Ni2+、Zn2+、E-64、PMSF抑制其活性，EDTA、L-半胱氨酸、DTT均可增强其活性。通过SDS-PAGE检测rTsCL对天然蛋白的水解作用，rTsCL在酸性条件下降解Hb（人、小鼠、猪和牛）、牛血清白蛋白和人血清白蛋白，推测其在旋毛虫体内参与了营养物质的降解；rTsCL降解细胞外基质蛋白（纤连蛋白、胶原蛋白I和层粘连蛋白），表明其在旋毛虫侵入宿主肠上皮细胞过程中发挥了重要的功能；分析rTsCL与宿主免疫球蛋白的相互作用发现， rTsCL降解小鼠IgG、人IgG和IgM，帮助旋毛虫逃避宿主的免疫应答。rTsCL对天然底物蛋白的水解作用可以被E64完全抑制。将rTsCL蛋白肌肉注射免疫小鼠，感染旋毛虫后，成虫与肌肉虫的减虫率高于标签组与佐剂组。该项目对于阐明旋毛虫免疫逃避机制具有重要的意义，并为研制抗旋毛虫病疫苗奠定了基础。