旋毛虫新型天冬氨酸蛋白酶-1 的分子特征。

Molecular characterization of a novel aspartyl protease-1 from Trichinella spiralis.

机构信息

Department of Parasitology, Medical College, Zhengzhou University, Zhengzhou 450052, PR China.

出版信息

Res Vet Sci. 2021 Jan;134:1-11. doi: 10.1016/j.rvsc.2020.11.008. Epub 2020 Nov 27.

DOI:10.1016/j.rvsc.2020.11.008

Abstract

The aim of this study was to characterize the biological properties of a novel aspartic protease-1 from Trichinella spiralis (TsASP1) and evaluate its potential in inducing immune response. TsASP1 gene was cloned and expressed in Escherichia coli BL21 (DE3). On Western blotting analysis with anti-rTsASP1 serum, native TsASP1 was detected in various T. spiralis phases other than newborn larvae (NBL). qPCR results showed that TsASP1 transcription was the highest in intestinal infective larvae (IIL) and the lowest in the NBL stage. Immunofluorescence test result shows that native TsASP1 was principally localized in stichosome, muscle cells of muscle larvae (ML) and IIL, and surrounded intrauterine embryos in female adult worms (AW). After silencing TsASP1 gene of the ML by siRNA, the worm development was significantly inhibited, showed by shorter AW and more wrinkles and longitudinal crack on epicuticle of AW on scanning electron microscopy; the AW and ML burdens were reduced by 41.82 and 56.36% respectively, compared with the control siRNA or PBS group (P < 0.001). Immunization of mice with rTsASP1 elicited an evident antibody response (serum IgG, IgG1/IgG2a and enteral sIgA), and systemic (spleen) and intestinal local mucosal (mesenteric lymph node) cellular immune response, demonstrated by a prominent elevation of IFN-γ and IL-4. The results suggested TsASP1 participated in T. spiralis development and survival in host, and immunization of mice with rTsASP1 induced systemic/intestinal local mucosal humoral and cellular immune response against Trichinella.

摘要

本研究旨在描述旋毛虫新型天冬氨酸蛋白酶-1（TsASP1）的生物学特性，并评估其诱导免疫反应的潜力。TsASP1 基因在大肠杆菌 BL21（DE3）中进行克隆和表达。用抗 rTsASP1 血清进行 Western blot 分析，在新生幼虫（NBL）以外的旋毛虫各期均检测到天然 TsASP1。qPCR 结果显示，TsASP1 转录在肠感染性幼虫（IIL）中最高，在 NBL 阶段最低。免疫荧光试验结果表明，天然 TsASP1 主要定位于肌幼虫（ML）和 IIL 的肌束和肌细胞中，并在雌性成虫（AW）的子宫内胚胎周围。用 siRNA 沉默 ML 中的 TsASP1 基因后，AW 的发育受到明显抑制，表现在 AW 的扫描电子显微镜下的外皮上皱纹和纵向裂纹增多，AW 和 ML 的负荷分别减少了 41.82%和 56.36%，与对照 siRNA 或 PBS 组相比（P<0.001）。用 rTsASP1 免疫小鼠可引起明显的抗体反应（血清 IgG、IgG1/IgG2a 和肠内 sIgA），以及全身（脾脏）和肠道局部黏膜（肠系膜淋巴结）细胞免疫反应，表现为 IFN-γ 和 IL-4 的显著升高。结果表明 TsASP1 参与旋毛虫在宿主中的发育和存活，rTsASP1 免疫小鼠可诱导针对旋毛虫的全身/肠道局部黏膜体液和细胞免疫反应。