旋毛虫Nudix 水解酶TsNd在幼虫侵入肠黏膜中的作用及机制研究

Trichinella spiralis is a foodborne zoonotic parasite. Once ingested, the critical process for establishing Trichinella infection is whether the larvae penetrate intestinal mucosa to develop further or are expulsed from the intestines. The larval invasion of intestinal mucosa is not only the result of a simple mechanical penetration, as the larvae do not possess oral appendices or a spike. Our previous studies demonstrated that T. spiralis Nudix hydrolase (TsNd) was secreted by the larvae co-cultured with intestinal epithelial cells (IECs) and entered into the IECs. The mice immunized with T7 Phage-displayed TsNd polypeptides showed a 62.8% reduction in adult worms following challenge with T. spiralis muscle larvae, suggesting that the larval invasion of IECs might be mediated by TsNd. However, to date, the functions of TsNd, the interactions between TsNd and host IECs, and the mechanism are unknown. The TsNd genes will be cloned, expressed and purified. The antigenicity and enzymatic activity of recombinant TsNd (rTsNd) protein will be identified by Western blot, ELISA and biochemical methods. The specific binding of recombinant TsNd with IECs will be confirmed by immunohistochemical method (e.g. immunofluorescence test, IFT). The promotion or suppression and blocking of recombinant TsNd and anti- rTsNd antibodies for the larval invasion of IEC and intestinal mucosa will be observed by using the in vitro cell model and in animal experiment. The worm expulsion in intestine of the mice immunized with recombinant TsNd will be also evaluated. Finally, the affect of blocking of the worm TsNd for the larval invasion of TsNd and intestinal mucosa is investigated. This project will elucidate the role of TsNd in the invasion of intestinal mucosa by T. spiralis and its mechanism. It will also lay the foundation for developing the trichinellosis vaccine with high protective effects (the ingested T. spiralis larvae will be eliminated in the intestine stage) and studying the new drugs against Trichinella.

旋毛虫是一种食源性人兽共患寄生虫，幼虫被摄入后侵入肠粘膜继续发育或从肠道排出是能否感染宿主的关键。由于幼虫口孔无齿状和矛状结构，幼虫侵入肠粘膜不是简单的机械性损伤。我们的研究发现，与肠上皮细胞（IECs）共培养后幼虫分泌的Nudix 水解酶（TsNd）可进入IECs内；在T7噬菌体展示的TsNd 多肽免疫小鼠，肠道成虫减少62.8%，提示TsNd可能介导了幼虫对IECs的侵入，但目前对TsNd的功能、与IECs的相互作用及机制均不清楚。本项目将克隆表达TsNd，应用RNAi明确TsNd基因功能，IFA等鉴定重组TsNd 与IECs结合的特异性；体外与动物实验观察重组TsNd 及抗重组TsNd抗体对幼虫侵入IECs与肠粘膜及虫体发育的影响以及肠道排虫反应。该项目将阐明TsNd在旋毛虫侵入肠粘膜中的作用及机制，并为研制高保护性的旋毛虫病疫苗（将旋毛虫消灭在肠道期）及抗旋毛虫新药等奠定基础。

旋毛虫是一种重要的食源性人兽共患寄生虫，但旋毛虫对肠黏膜的侵入机制尚不清楚。Nudix 水解酶（TsNd）可能介导了幼虫对肠上皮细胞（IECs）的侵入。该项目的主要研究内容是明确TsNd的生物学特性、功能及其与IECs的相互作用等。该项目克隆与表达出了具有Nudix水解酶活性的旋毛虫TsNd。qPCR、Western blot与IFT结果显示，TsNd在旋毛虫肌幼虫（ML）、肠道感染性幼虫（IIL）、成虫和新生幼虫（NBL）期均有表达，在IIL期的表达水平较高；TsNd主要定位在虫体皮层与杆状体。Far-Western、IFT及共聚焦显微镜检查结果表明，rTsNd能与IECs膜特异性结合。rTsNd对旋毛虫幼虫侵入IECs具有促进作用，而抗rTsNd抗体对旋毛虫侵入IECs具有明显的抑制与部分阻断作用，且呈抗体剂量依赖性。抗rTsNd抗体依赖的ADCC在体外对旋毛虫幼虫具有杀伤作用。RNA干扰TsNd基因可明显抑制幼虫对IECs的侵入、降低幼虫对小鼠的感染性以及幼虫在小鼠体内的发育与存活。rTsNd 蛋白免疫小鼠诱导了Th2型为主的免疫应答，攻击感染后3d与35d肠道成虫和肌幼虫减虫率分别为57.70%和56.90%，明显高于标签蛋白与佐剂对照组（P<0.01）。将pcDNA3.1-TsNd肌肉注射小鼠或经口免疫小鼠，可诱导特异性的肠道分泌型IgA及Th1/Th2混合型免疫应答；肌肉注射组的肠道成虫和肌幼虫减虫率分别为40.44%和53.9%，口服免疫组的成虫和肌幼虫减虫率分别为73.32%和49.5%，结果表明TsNd DNA疫苗肌肉注射与口服免疫小鼠均可产生部分免疫保护作用，口服组的成虫减虫率明显高于肌肉注射组（P<0.01）。该项目为进一步研制抗肠道期旋毛虫疫苗及抗旋毛虫新药等奠定了基础。