Sánchez-Montejo Javier, Strilets Tania, Manzano-Román Raúl, López-Abán Julio, García-Blanco Mariano A, Vicente Belén, Muro Antonio
Infectious and Tropical Diseases Research Group (e-INTRO), Biomedical Research Institute of Salamanca Research Centre for Tropical Diseases at the University of Salamanca (IBSAL-CIETUS), 37007 Salamanca, Spain.
Infectious Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.
Int J Mol Sci. 2025 Jan 30;26(3):1190. doi: 10.3390/ijms26031190.
is a parasitic trematode responsible for fascioliasis, a significant zoonotic disease affecting livestock worldwide, as well as humans. This study identifies peptides with potential for use in vaccines against and validates multi-epitope constructs from those peptides in vitro. Putative protein sequences derived from the genome of were integrated with phase-specific transcriptomic data to prioritize highly expressed proteins. Among these, extracellular proteins were selected using DeepLoc 2.0 and strong binding affinities across diverse human and murine alleles were predicted with the IEDB MHC II tool. Peptides were further selected based on their toxicity, immunogenicity, and allergenicity. Finally, 55 high-priority candidates were obtained. To express these candidates, mRNA constructs encoding various combinations of these peptides were designed, synthesized using in vitro transcription with T7 or SP6 RNA polymerases, and transfected into cells for expression analysis. SP6 polymerase produced proper capping using CleanCapAG and was far superior in transcribing peptide constructs. Peptides fused in frame with eGFP were expressed efficiently, particularly when peptides were positioned at the 3' terminus, opening a new field of peptide vaccines created using mRNA technology.
是一种寄生性吸虫，可引发肝片吸虫病，这是一种影响全球牲畜以及人类的重要人畜共患病。本研究鉴定了具有用于抗疫苗潜力的肽，并在体外验证了来自这些肽的多表位构建体。从的基因组中获得的推定蛋白质序列与阶段特异性转录组数据整合，以对高表达蛋白质进行优先级排序。其中，使用DeepLoc 2.0选择细胞外蛋白质，并使用IEDB MHC II工具预测其在不同人类和鼠类等位基因上的强结合亲和力。肽进一步根据其毒性、免疫原性和致敏性进行选择。最后，获得了55个高优先级候选物。为了表达这些候选物，设计了编码这些肽各种组合的mRNA构建体，使用T7或SP6 RNA聚合酶通过体外转录合成，并转染到细胞中进行表达分析。SP6聚合酶使用CleanCapAG产生了合适的帽结构，并且在转录肽构建体方面远优于其他酶。与eGFP框内融合的肽能够高效表达，特别是当肽位于3'末端时，这开辟了使用mRNA技术创建肽疫苗的新领域。
