Department of Parasitology, Medical College, Zhengzhou University, 40 Daxue Road, Zhengzhou 450052, People's Republic of China.
School of Life Science, Zhengzhou University, 40 Daxue Road, Zhengzhou 450052, People's Republic of China.
Trop Biomed. 2021 Mar 1;38(1):160-171. doi: 10.47665/tb.38.1.028.
Trichinella spiralis is an important foodborne zoonotic parasite and it is necessary to develop vaccine to prevent T. spiralis infection in food animals. T. spiralis aspartic protease-2 (TsASP2) has been demonstrated to play a crucial role in larval invasion of intestinal epithelium cells (IECs). The purpose of this study was to assess the interaction between TsASP2 and IECs and to investigate the immune protection elicited by vaccination with rTsASP2. The results showed that the enzymatic activity of native aspartic protease was detected in crude proteins of all T. spiralis development stages other than NBL stage, the highest activity was observed in the IIL stage. The results of Western blot showed that TsASP2 protein was expressed at ML, IIL and AW but not NBL, and the TsASP2 expression level at IIL stage was significantly higher than those of other three worm stages (P < 0.05). The specific binding between rTsASP2 and IECs was observed by immunofluorescence test (IFT) and confocal microscopy, and the binding site was localized at the IEC membrane and this binding ability was inhibited by aspartic protease specific inhibitor pepstain A. The results of ELISA showed that the binding ability was protein dose-dependent. Vaccination with rTsASP2 triggered a mixed Th1/Th2 humoral and mucosal immune responses, as demonstrated by the elevation levels of Th1/Th2 cytokines (IFN-γ and IL-4) secreted by the spleen and mesenteric lymph nodes (MLNs) of immunized mice. The mice vaccinated with rTsASP2 exhibited a 54.17% reduction in enteral adult worms and a 54.58% reduction in muscle larvae after T. spiralis challenge. The results demonstrated that TsASP2 might be a potential molecular target for anti-Trichinella vaccines.
旋毛虫是一种重要的食源性人畜共患寄生虫，有必要开发疫苗来预防食源性动物感染旋毛虫。旋毛虫天冬氨酸蛋白酶-2（TsASP2）已被证明在幼虫侵入肠上皮细胞（IEC）中发挥关键作用。本研究旨在评估 TsASP2 与 IEC 之间的相互作用，并研究用 rTsASP2 疫苗接种引发的免疫保护作用。结果表明，在除 NBL 期外的所有旋毛虫发育阶段的粗蛋白中均检测到天然天冬氨酸蛋白酶的酶活性，在 IIL 期观察到最高活性。Western blot 结果显示，TsASP2 蛋白在 ML、IIL 和 AW 期表达，但在 NBL 期不表达，且在 IIL 期的表达水平明显高于其他三个虫期（P<0.05）。免疫荧光试验（IFT）和共聚焦显微镜观察到 rTsASP2 与 IEC 之间的特异性结合，结合部位定位于 IEC 膜，该结合能力被天冬氨酸蛋白酶特异性抑制剂 pepstatin A 抑制。ELISA 结果表明，结合能力呈蛋白剂量依赖性。rTsASP2 疫苗接种引发了混合 Th1/Th2 体液和黏膜免疫反应，这表现为免疫小鼠的脾和肠系膜淋巴结（MLN）分泌的 Th1/Th2 细胞因子（IFN-γ 和 IL-4）水平升高。rTsASP2 疫苗接种的小鼠在旋毛虫攻毒后肠道成虫减少了 54.17%，肌肉幼虫减少了 54.58%。结果表明，TsASP2 可能是抗旋毛虫疫苗的潜在分子靶标。
