旋毛虫发育过程中蜕皮蛋白酶的鉴定及作用机制

Molting is the characteristic landmark in the process of larval development in nematodes. The larvae cannot develop further if the molting could not be completed. Trichinella spiralis muscle larvae are activated to the intestinal infective larvae (IIL) in the host’s intestine, and become adult worm after undergoing four molts within 31 hours after infection. Our previous studies indicated that the IIL invaded intestinal epithelial cells (IECs) monolayer in vitro, migrated, molt, developed in cell monolayer, and produced some proteases. But, it is unclear which proteases play the essential role during the larval molting and their functions. In this project, the T. spiralis molting proteases in molting fluid and somatic proteins of IIL1-IIL4 will be screened and identified by proteomics and enzymology. After being cloned and expressed, the expression and location of the molting proteases in various developmental stages, and their enzyme activity will be assayed and characterized by qPCR, Western blot, IFT and other methods. The role and function of these molting proteases in the process of larval molting will be confirmed by specific antibody blockage, enzyme inhibitors and RNAi in the in vitro invasion model of T. spiralis and animal experiment. The molting protease hydrolysis or degradation of different proteins and surface proteins of T. spiralis larvae will be investigated, and the immune protection against T. spiralis infection will be also evaluated in a model of mice vaccinated with the proteases. This project will elucidate the function and action mechanism of T. spiralis molting proteases, and lay the foundation to develop the vaccine against the early IIL stage of T. spiralis and the molecular target of new drugs against IIL stages.

蜕皮是线虫幼虫发育过程中的一个标志性事件；如果不能完成蜕皮，则幼虫不能进一步发育。旋毛虫肌幼虫在宿主小肠内被激活为肠道感染性幼虫（IIL），感染后31h经4次蜕皮发育为成虫。我们前期研究发现，IIL可在体外侵入肠上皮细胞（IECs）单层、并在其中移行、蜕皮发育及产生一些蛋白酶，但哪些蛋白酶在幼虫蜕皮时发挥了主要作用及功能则不清楚。本项目将应用蛋白组学与酶学从IIL1~IIL4蜕皮液与虫体蛋白中鉴定旋毛虫蜕皮蛋白酶，克隆表达后应用qPCR、Western blot、IFT等检测其表达时相、定位及酶活性；通过旋毛虫-IECs体外侵入模型及动物实验，应用抗体封闭、酶抑制剂及RNAi鉴定蛋白酶在幼虫蜕皮中的作用与功能；观察蛋白酶对不同蛋白与旋毛虫表面蛋白的水解作用及免疫小鼠的保护作用。该项目将阐明旋毛虫蜕皮蛋白酶的功能及作用机制、为筛选抗发育早期（IIL期）旋毛虫疫苗与新药的靶分子等奠定基础。

蜕皮是线虫幼虫发育过程中的一个标志性事件；如果不能完成蜕皮，则幼虫不能进一步发育。旋毛虫肌幼虫在宿主小肠内被激活为肠道感染性幼虫（IIL），感染后31 h经4次蜕皮发育为成虫。我们前期研究发现，IIL可在体外侵入肠上皮细胞（IEC）单层、并在其中移行、蜕皮发育及产生一些蛋白酶，但哪些蛋白酶在旋毛虫幼虫蜕皮时发挥了主要作用则不清楚。该项目通过无标记定量蛋白质组学、酶抑制剂、底物、明胶酶谱与质谱分析等方法，鉴定出了旋毛虫IIL的蜕皮相关蛋白酶包括半胱氨酸蛋白酶、金属蛋白酶、天冬氨酸蛋白酶、谷氨酰胺合成酶、丝氨酸蛋白酶等。该项目克隆表达了旋毛虫无机焦磷酸酶（TsPPase）、金属蛋白酶（Tsdpy31）、天冬氨酸蛋白酶1（TsASP1）、谷氨酰胺合成酶（TsGS）及丙酮酸激酶（TsPKM）等，研究了其生物学特性与功能，发现上述蛋白酶在旋毛虫IIL期的表达水平明显高于其他虫期，主要定位在皮层与杆状体。通过体外侵入模型及动物实验，应用重组蛋白酶、特异性抗体、酶抑制剂或RNAi，发现上述蛋白酶在旋毛虫IIL蜕皮与发育过程中发挥了重要作用。TsPPase特异性siRNA在体外与体内对IIL蜕皮的抑制率分别为47.32%与34%（P < 0.05）。rTsdpy31具有天然酶活性，能够与旋毛虫皮层的胶原蛋白rTscol2特异性结合，特异性dsRNA增加了虫体皮层的通透性，rTsdpy31参与了旋毛虫蜕皮过程中新皮层形成与旧皮层剥离。TsASP1特异性siRNA导致了虫体表皮出现显著皱褶。沉默TsGS基因使IIL蜕皮率降低了47.24%（P < 0.01）。沉默TsPKM使IIL蜕皮率下降了8.67%（P < 0.05）。rTsPPase与rTsASP1免疫小鼠诱导了明显的保护性免疫。结果表明这些蛋白酶参与了旋毛虫的蜕皮与发育，可作为抗旋毛虫IIL期疫苗与新药的侯选靶分子。