The critical step of Trichinella spiralis infection is that the muscle larvae (ML) are activated to intestinal infective larvae (IIL) which invade the intestinal columnar epithelium to further develop. The IIL excretory/secretory (ES) proteins play an important role in host-parasite interaction. Proteolytic enzymes are able to mediate the tissue invasion, thereby increasing the susceptibility of parasites to their hosts. The aim of the current study was to screen and identify the natural active proteases in T. spiralis IIL ES proteins using Western blot and gel zymography combined with liquid chromatography tandem mass spectrometry (LC-MS/MS). The T. spiralis ML and IIL ES proteins were collected from the in vitro cultures and their enzymatic acitvities were examined by gelatin zymography and azocasein degradation. The protease activities were partially inhibited by PMSF, E-64 and EDTA. Three protein bands (45, 118 and 165 kDa) of T. spiralis IIL ES proteins were identified by shotgun LC-MS/MS because they have hydrolytic activity to gelatin compared to the ML ES proteins. Total of 30 T. spiralis proteins were identified and they are mainly serine proteinases (19), but also metalloproteinases (7) and cysteine proteinases (3). The qPCR results indicated that transcription levels of four T. spiralis protease genes (two serine proteases, a cathepsin B-like cysteine proteinase and a zinc metalloproteinase) at IIL stage were obviously higher than at the ML stage. These proteolytic enzymes are directly exposed to the host intestinal milieu and they may mediate the worm invasion of enteral epithelium and escaping from the host's immune responses. The results provide the new insights into understanding of the interaction of T. spiralis with host and the invasion mechanism.

旋毛虫旋毛虫感染的关键步骤是将肌肉幼虫（ML）激活为肠道感染性幼虫（IIL），后者会侵入肠道柱状上皮进一步发展。IIL分泌/分泌（ES）蛋白在宿主-寄生虫相互作用中起重要作用。蛋白水解酶能够介导组织入侵，从而增加了寄生虫对其宿主的敏感性。本研究的目的是使用蛋白质印迹和凝胶酶谱结合液相色谱串联质谱法（LC-MS / MS）筛选和鉴定螺旋藻IIL ES蛋白中的天然活性蛋白酶。的旋毛虫ML和IIL ES蛋白从所收集的体外通过明胶酶谱法和偶氮酪蛋白降解检查培养物及其酶活性。蛋白酶活性被PMSF，E-64和EDTA部分抑制。通过shot弹枪LC-MS / MS鉴定了螺旋旋毛虫ILS ES蛋白的三个蛋白带（45、118和165 kDa），因为与ML ES蛋白相比，它们对明胶具有水解活性。总共鉴定出30种螺旋螺旋体蛋白，它们主要是丝氨酸蛋白酶（19），但也包括金属蛋白酶（7）和半胱氨酸蛋白酶（3）。qPCR结果表明四个螺旋藻的转录水平IIL期的蛋白酶基因（两种丝氨酸蛋白酶，组织蛋白酶B样半胱氨酸蛋白酶和锌金属蛋白酶）明显高于ML期。这些蛋白水解酶直接暴露于宿主肠道环境，它们可能介导蠕虫入侵肠上皮并逃脱宿主的免疫反应。结果为了解螺旋螺旋体与宿主的相互作用和入侵机制提供了新的见解。