旋毛虫成虫侵入蛋白的筛选、鉴定及作用机制

Trichinella spiralis larvae grow to adult worms in intestine, and then invade intestinal mucosa to further develop and produce the newborn larvae. Adult is also the invasion and pathogenic stage of T. spiralis. Because the parasite do not possess oral appendices or a spike, the invasion of intestinal mucosa by T. spiralis adult is not the simple mechanical penetration, is most likely mediated by these proteases in excretory-secretory (ES) and surface proteins of adult worms, but the invasion mechanisms are unclear. In this project, the adult high-expressed proteins bound to intestinal epithelial cells (IEC) will be screened by immunoproteomics, cloned and expressed. The adult invasion proteins are screened by using the in vitro model of T. spiralis invasion of IEC established in our previous study and animal experiment. The specificity of invasion proteins binding to ICE will be identified by immunofluorescent test (IFT) and Far-Western analysis. The membrane proteins combined with invasion proteins are characterized by pull down and mass spectrometry. The funtions of invasion proteins at gene and protein levels will be investigated by RNA interference and enzyme hydrolysis assays. The regulation role of the invasion proteins on the intestinal worm expulsion and immune responses (IgG, sIgA and cytokines) will be evaluated in mice infected with T. spiralis. This project will elucidate the interaction between adult invasion proteins and host IEC, invasive and pathogenic mechanism of T. spiralis adults. It will be contributed to find the target molecular of anti-Trichinella adult drugs and studying the polyvalent vaccine against T. spiralis adult worms.

旋毛虫幼虫在小肠发育为成虫后再次侵入肠黏膜发育并产新生幼虫，成虫也是旋毛虫的侵入与致病期，由于口孔无齿状和矛状结构，成虫的侵入不是简单的机械性穿透，可能是其ES与表面蛋白中的一些蛋白酶介导的，但侵入机制尚不清楚。本项目将应用免疫蛋白组学从成虫ES和表面蛋白中筛选与肠上皮细胞（IEC）结合的成虫高表达的蛋白进行克隆表达，应用前期建立的旋毛虫－IEC体外侵入模型与动物实验筛选成虫侵入蛋白；通过IFT与Far-Western鉴定侵入蛋白与IEC结合的特异性，pull down与质谱分析鉴定侵入蛋白结合的IEC膜蛋白；应用RNAi及酶水解试验从基因与蛋白水平鉴定侵入蛋白的功能；研究侵入蛋白对肠道排虫与免疫反应（IgG与sIgA及细胞因子水平）的调节作用。该项目将阐明旋毛虫成虫侵入蛋白与宿主IEC相互作用及侵入和致病机制，为寻找抗旋毛虫成虫药物靶分子及研制抗旋毛虫成虫的多价疫苗等奠定基础。

旋毛虫幼虫在小肠发育为成虫后再次侵入肠黏膜发育并产新生幼虫，成虫也是旋毛虫的侵入期，但关于成虫的侵入蛋白及作用机制尚不清楚。该项目的主要研究内容是筛选旋毛虫成虫期高表达的蛋白，鉴定其生物学特性、功能及作用机制。该项目应用免疫蛋白组学从旋毛虫成虫ES蛋白中，筛选出了成虫侵入蛋白（TsDNase II-1/7、TsCP及TsSPI），进行了克隆表达及鉴定，发现这3种蛋白在旋毛虫成虫期的表达明显高于幼虫期，主要定位在虫体表皮、杆状体及胚胎等。通过旋毛虫－肠上皮细胞（IEC）体外侵入模型与动物实验，发现3种抗成虫侵入蛋白抗体，对旋毛虫侵入IEC与肠黏膜具有明显的抑制作用，并可抑制虫体在肠道内的发育与存活，这种抑制作用具有抗体剂量依赖性。应用Far-Western、IFA等发现rTsSPI能够与IEC特异性结合并进入胞浆内；rTsSPI具有抑制宿主胰蛋白酶活性的功能。RNAi沉默TsSPI基因明显降低了旋毛虫的感染性、抑制了旋毛虫对IEC及肠黏膜的侵入，以及虫体在小鼠肠道内的发育和生殖。将rTsDNase II-1/7、TsCP及TsSPI免疫小鼠后，均诱导产生了以Th2型为主的免疫应答，对旋毛虫攻击感染产生了明显的免疫保护，免疫小鼠的成虫减虫率分别为40.36%/34.86%，54.29%及62.2%；将rTsDNase II-1/7 DNA疫苗经口免疫接种小鼠后，产生了明显的肠道黏膜免疫应答与系统的Th1/Th2混合型免疫应答，以及显著的免疫保护，成虫和肌幼虫减虫率分别为53.85%/46.15%和59.26%/53.41%。抗TsDNase II-1/7、TsCP及TsSPI抗体依赖的ADCC对旋毛虫具有特异性杀伤作用。结果表明，TsDNase II-1/7、TsCP及TsSPI是旋毛虫成虫对宿主肠黏膜的侵入蛋白，可作为研制抗旋毛虫成虫药物与多价疫苗候选的分子靶标。