School of Public Health, Shantou University, Shantou, China.
College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, China.
Front Cell Infect Microbiol. 2021 Oct 18;11:779259. doi: 10.3389/fcimb.2021.779259. eCollection 2021.
Clonorchiasis, which is caused by , is an important foodborne disease worldwide. The excretory-secretory products (ESPs) of play important roles in host-parasite interactions by acting as causative agents. In the present study, the ESPs and sera positive for were collected to identify proteins specific to the sera of (i.e., proteins that do not cross-react with and ) at different infection periods. Briefly, white Japanese rabbits were artificially infected with , and their sera were collected at 7 days post-infection (dpi), 14 dpi, 35 dpi, and 77 dpi. To identify the specific proteins in , a co-immunoprecipitation (Co-IP) assay was conducted using shotgun liquid chromatography tandem-mass spectrometry (LC-MS/MS) to pull down the sera roots of , , and . For the annotated proteins, 32, 18, 39, and 35 proteins specific to were pulled down by the infected sera at 7, 14, 35, and 77 dpi, respectively. Three proteins, Dynein light chain-1, Dynein light chain-2 and Myoferlin were detected in all infection periods. Of these proteins, myoferlin is known to be overexpressed in several human cancers and could be a promising biomarker and therapeutic target for cancer cases. Accordingly, this protein was selected for further studies. To achieve a better expression, myoferlin was truncated into two parts, Myof1 and Myof2 (1,500 bp and 810 bp), based on the antigenic epitopes provided by bioinformatics. The estimated molecular weight of the recombinant proteins was 57.3 ku (Myof1) and 31.3 ku (Myof2). Further, both Myof1 and Myof2 could be probed by the sera from rabbits infected with . No cross-reaction occurred with the positive sera of , , and negative controls. Such findings indicate that myoferlin may be an important diagnostic antigen present in the ESPs. Overall, the present study provides new insights into proteomic changes between ESPs and hosts in different infection periods by LC-MS/MS. Moreover, myoferlin, as a biomarker, may be used to develop an objective method for future diagnosis of clonorchiasis.
肝吸虫病是由肝吸虫引起的，是一种重要的食源性疾病。肝吸虫的排泄-分泌产物（ESPs）在宿主-寄生虫相互作用中发挥重要作用，作为致病因子。在本研究中，收集了肝吸虫的 ESPs 和血清阳性产物，以鉴定在不同感染期对肝吸虫血清特异的蛋白（即与华支睾吸虫和异形异形吸虫不发生交叉反应的蛋白）。简要地说，用人工感染白日本兔，在感染后 7 天（dpi）、14 dpi、35 dpi 和 77 dpi 收集血清。为了鉴定肝吸虫中的特异蛋白，使用免疫共沉淀（Co-IP）测定结合液相色谱串联质谱（LC-MS/MS）下拉感染血清的虫体根、华支睾吸虫和异形异形吸虫。对于注释蛋白，在感染后 7、14、35 和 77 dpi 时，分别有 32、18、39 和 35 个蛋白被感染血清下拉。三种蛋白，即动力蛋白轻链-1、动力蛋白轻链-2 和肌球蛋白，在所有感染期均被检测到。其中，肌球蛋白已被证实在几种人类癌症中过度表达，可能是癌症病例的一个有前途的生物标志物和治疗靶点。因此，选择该蛋白进行进一步研究。为了获得更好的表达，根据生物信息学提供的抗原表位，将肌球蛋白截断为两部分，Myof1 和 Myof2（1500 bp 和 810 bp）。重组蛋白的估计分子量为 57.3 ku（Myof1）和 31.3 ku（Myof2）。此外，Myof1 和 Myof2 都可以被感染兔的血清探测到。与阳性血清的交叉反应未发生华支睾吸虫、异形异形吸虫和阴性对照。这些发现表明肌球蛋白可能是 ESPs 中一种重要的诊断抗原。总的来说，本研究通过 LC-MS/MS 提供了在不同感染期 ESPs 和宿主之间蛋白质组变化的新见解。此外，肌球蛋白作为一种生物标志物，可能用于开发未来肝吸虫病的客观诊断方法。
