Institute of Biological, Environmental and Rural Sciences (IBERS), Edward Llwyd Building, Aberystwyth University, UK.
Vet Parasitol. 2010 Apr 19;169(1-2):62-75. doi: 10.1016/j.vetpar.2009.12.031. Epub 2010 Jan 4.
Fasciola hepatica is responsible for human disease and economic livestock loss on a global scale. We report the first post-genomic investigation of cellular proteins expressed by embryonic F. hepatica via two-dimensional electrophoresis, image analysis and tandem mass spectrometry. Antioxidant proteins and protein chaperones are prominently expressed by embryonic F. hepatica. Molecular differences between the egg and other characterized F. hepatica lifecycle stages were noted. Furthermore, proteins expressed within liver fluke eggs differ to those isolated from the well-characterized eggs of the human blood flatworm Schistosoma mansoni were revealed. Plasticity in expression of major proteins, particularly a prominently expressed 65kDa protein cluster was seen between natural populations of embryonating F. hepatica eggs suggesting that liver fluke embryogenisis is a plastic process. Immunoblotting revealed that the abundant 65kDa protein cluster is recognised by infection sera from three F. hepatica challenged host species. Mass spectrometry and BLAST analyses demonstrated that the 65kDa antigen shows homology to egg antigens of other flatworm parasites, and is represented in a F. hepatica EST database constructed from adult fluke transcripts. EST clones encoding the egg antigen were re-sequenced, predicting two forms of the protein. Four clones predict a 312 aa polypeptide, three clones encode a putative 110 amino acid extension at the N-terminus which may be involved in protein secretion, although this extension was not expressed by natively extracted proteins. Consistent expression of alpha crystallin domains confirmed the protein to be a member of the alpha crystallin containing small heat shock protein (AC/sHSP) superfamily. AC/sHSPs are ubiquitous in nature, however, this is the first time a member of this protein superfamily has been described from F. hepatica. The antigenic AC/sHSP was named Fh-HSP35alpha based on predictions of molecular weight. Production of recombinant Fh-HSP35alpha reveals considerable mass discrepancy between native and recombinant proteins, although descriptions of other characterized flatworm AC/sHSPs, suggest that the native form is a dimer. Immunoblot analyses confirm that the recombinant protein is recognised by F. hepatica challenged hosts, but does not react with sera from non-infected animals. We discuss the potential of recombinant Fh-HSP35alpha as an egg-based diagnostic marker for liver fluke infection.
肝片形吸虫在全球范围内导致人类疾病和经济家畜损失。我们通过二维电泳、图像分析和串联质谱法报告了对胚胎肝片形吸虫表达的细胞蛋白的首次后基因组研究。抗氧化蛋白和蛋白伴侣在胚胎肝片形吸虫中表达明显。在卵和其他特征化的肝片形吸虫生命周期阶段之间注意到分子差异。此外，还揭示了在肝片形吸虫卵内表达的蛋白质与已表征的人类血吸虫曼氏血吸虫的卵中分离的蛋白质不同。在天然种群的胚胎肝片形吸虫卵中观察到主要蛋白质表达的可塑性，特别是一个表达明显的 65kDa 蛋白簇，表明肝片形吸虫胚胎发生是一个可塑性过程。免疫印迹显示，大量的 65kDa 蛋白簇被来自三种感染肝片形吸虫宿主物种的感染血清识别。质谱分析和 BLAST 分析表明，65kDa 抗原与其他扁形虫寄生虫的卵抗原具有同源性，并且在从成虫转录物构建的肝片形吸虫 EST 数据库中代表。编码卵抗原的 EST 克隆被重新测序，预测两种蛋白质形式。四个克隆预测一个 312 个氨基酸的多肽，三个克隆编码一个可能在蛋白质分泌中起作用的 N 端 110 个氨基酸的延伸，尽管这种延伸没有被天然提取的蛋白质表达。α 晶体结构域的一致表达证实该蛋白是含有小热休克蛋白 (AC/sHSP) 超家族的蛋白。AC/sHSP 在自然界中无处不在，但这是首次从肝片形吸虫中描述该蛋白超家族的成员。该抗原 AC/sHSP 根据分子量预测被命名为 Fh-HSP35alpha。重组 Fh-HSP35alpha 的产生揭示了天然和重组蛋白之间相当大的质量差异，尽管对其他特征化的扁形虫 AC/sHSP 的描述表明，天然形式是二聚体。免疫印迹分析证实重组蛋白被感染肝片形吸虫的宿主识别，但不与未感染动物的血清反应。我们讨论了重组 Fh-HSP35alpha 作为肝片形吸虫感染基于卵的诊断标志物的潜力。
