National Reference Laboratory for Animal Schistosomiasis; Key Laboratory of Animal Parasitology of Ministry of Agriculture and Rural Affairs, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
National Reference Laboratory for Animal Schistosomiasis; Key Laboratory of Animal Parasitology of Ministry of Agriculture and Rural Affairs, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China; Laboratory of Environmental Entomology, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China.
Acta Trop. 2022 Aug;232:106519. doi: 10.1016/j.actatropica.2022.106519. Epub 2022 May 16.
The tegument of schistosomes is the interface between the worm and the host environment. Some molecules distributed on the tegument participate in host-parasite interactions. Aspartyl aminopeptidase (AAP), identified on the tegument of Schistosoma japonicum (S. japonicum), facilitate protein turnover by acting in concert with other aminopeptidases. In this study, the gene encoding S. japonicum aspartyl aminopeptidase (SjAAP) was cloned, expressed and characterized. Quantitative real-time PCR analysis showed that SjAAP was expressed in all studied developmental stages. The transcript level was higher in 8, 14, 21, and 28 days old worms than the other detected stages. Moreover, the level of expression in 42-day-old male worms was significantly higher than that in females. The recombinant SjAAP (rSjAAP) was expressed as both supernatant and inclusion bodies in Escherichia coli BL21 cells. The enzymatic activity of rSjAAP was 4.45 U/mg. The Km and Vmax values for H-Asp-pNA hydrolysis were discovered to be 5.93 mM and 0.018 mM·min. Immunofluorescence analysis revealed that SjAAP is primarily distributed on the tegument and parenchyma of schistosomes. Western blot showed that rSjAAP possessed good immunogenicity. Although specific antibodies were produced in BALB/c mice vaccinated with rSjAAP emulsified with ISA 206 adjuvant, no significant reduction of worm burden and number of eggs in the liver was observed. Therefore, rSjAAP may not be suitable to act as a potential vaccine candidate against schistosomiasis japonica in mice. However, this study provides some foundation for further exploration of the biological function of this molecule.
血吸虫的表皮是虫体与宿主环境的界面。分布在表皮上的一些分子参与宿主-寄生虫相互作用。天冬氨酰氨基肽酶（AAP）在日本血吸虫（S. japonicum）的表皮上被识别出来，通过与其他氨基肽酶协同作用，促进蛋白质周转。在这项研究中，克隆、表达和表征了编码日本血吸虫天冬氨酰氨基肽酶（SjAAP）的基因。定量实时 PCR 分析表明，SjAAP 在所有研究的发育阶段都有表达。8、14、21 和 28 日龄虫体的转录水平高于其他检测阶段。此外，42 日龄雄性虫体的表达水平明显高于雌性。重组 SjAAP（rSjAAP）在大肠杆菌 BL21 细胞中以上清液和包涵体的形式表达。rSjAAP 的酶活性为 4.45 U/mg。发现 H-Asp-pNA 水解的 Km 和 Vmax 值分别为 5.93 mM 和 0.018 mM·min。免疫荧光分析表明 SjAAP 主要分布在血吸虫的表皮和实质中。Western blot 显示 rSjAAP 具有良好的免疫原性。尽管用 ISA 206 佐剂乳化的 rSjAAP 接种 BALB/c 小鼠后产生了特异性抗体，但未观察到对虫体负荷和肝脏中虫卵数量的显著减少。因此，rSjAAP 可能不适合作为日本血吸虫的潜在疫苗候选物。然而，本研究为进一步探索该分子的生物学功能提供了一些基础。
