背景:一些研究报道了旋毛虫胞外囊泡在免疫调节和病原体诊断中的作用。目前,旋毛虫肌幼虫排泄/分泌产物(Ts-ML-ES)是国际旋毛虫病委员会(ICT)推荐的用于旋毛虫病血清学诊断的抗原。然而,它只能用于检测感染的中晚期,并与其他寄生虫检测发生交叉反应。因此,需要鉴定用于特异性检测早期旋毛虫病的抗原。
方法:通过超速离心分离旋毛虫肌幼虫(Ts-ML-EV)的胞外囊泡,并通过透射电子显微镜进行表征,纳米粒子跟踪分析,流式细胞术和蛋白质印迹。通过LC-MS/MS蛋白质组学分析Ts-ML-EV蛋白质谱以鉴定潜在抗原(Ts-TTPA)。将Ts-TTPA克隆到pMAL-c5X载体中,并表达为重组蛋白,以通过蛋白质印迹和ELISA评估检测到的抗原的潜力。
结果:隔离的Ts-ML-EV呈圆形或椭圆形(直径在110.1和307.6nm之间),显示双层膜结构。Ts-ML-EV上的特定表面标记是CD81、CD63、烯醇化酶和14-3-3蛋白。通过LC-MS/MS共鉴定出53种蛋白质。包括已报道作为潜在检测和疫苗候选物的各种分子。本研究中选择的Ts-TTPA的cDNA总长度为1152bp,编码384个氨基酸,分子量为44.19kDa。它含有胰蛋白酶结构域并且可以被抗His抗体识别。它在感染后15、25、35和60天(dpi)与感染了10,000个旋毛虫的猪血清反应。在10μg/ml时,该抗原可以在13dpi时检测到猪血清中的旋毛虫抗体。与感染其他寄生虫包括华支睾吸虫的猪血清没有交叉反应,弓形虫,猪带虫,蛔虫猪和毛虫猪。
结论:这项研究鉴定了潜在的早期检测抗原,并更彻底地表征了含丝氨酸蛋白酶结构域的蛋白质。细胞外囊泡蛋白可作为旋毛虫病早期检测的有效抗原。
BACKGROUND: Several studies have reported the roles of
Trichinella spiralis extracellular vesicles in immune regulation and pathogen diagnosis. Currently, the T. spiralis muscle larvae excretory/secretory product (Ts-ML-ES) is the antigen recommended by the International Commission on Trichinellosis (ICT) for serological diagnosis of trichinellosis. However, it can only be used to detect middle and late stages of infections, and cross-reactions with other parasite detections occur. Therefore, there is a need to identify antigens for specific detection of early stage trichinellosis.
METHODS: Extracellular vesicles of T. spiralis muscle larvae (Ts-ML-EVs) were isolated by ultracentrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis, flow cytometry and western blot. Ts-ML-EVs protein profiles were analyzed by LC-MS/MS proteomics for identification of potential antigens (Ts-TTPA). Ts-TTPA were cloned into pMAL-c5X vector and expressed as recombinant proteins for evaluation of potential as detected antigens by western blot and ELISA.
RESULTS: Isolated Ts-ML-EVs were round or elliptic (with diameters between 110.1 and 307.6 nm), showing a bilayer membrane structure. The specific surface markers on the Ts-ML-EVs were CD81, CD63, enolase and the 14-3-3 protein. A total of 53 proteins were identified by LC-MS/MS, including a variety of molecules that have been reported as potential detection and vaccine candidates. The cDNA of Ts-TTPA selected in this study has a total length of 1152 bp, encoding 384 amino acids with a molecular weight of 44.19 kDa. It contains a trypsin domain and can be recognized by anti-His antibody. It reacted with swine sera infected with 10,000 T. spiralis at 15, 25, 35 and 60 days post-infection (dpi). At 10 μg/ml, this antigen could detect T. spiralis antibodies from the swine sera at 13 dpi. There were no cross-reactions with the swine sera infected with other parasites including Clonorchis sinensis, Toxoplasma gondii, Taenia suis, Ascaris suis and Trichuris suis.
CONCLUSIONS: This study identifies potential early stage detection antigens and more thoroughly characterizes a serine protease domain-containing protein. Extracellular vesicle proteins may be explored as effective antigens for the early stage detection of trichinellosis.