Cloning, bioinformatics analysis, and expression of the ubiquitin 2 (ubq-2) gene from the dog roundworm Toxocara canis

Toxocariasis, caused by the dog roundworm <i>Toxocara canis,</i> is a globally distributed zoonotic parasitic disease that poses a significant threat to veterinary and public health. The ubiquitin plus an L40 ribosomal protein (UBQ-2) in parasites plays a crucial role in protein degradation and meiotic divisions, thereby affecting parasite development, survival, and parasite-host interactions. In this study, we identified for the first time a full-length cDNA encoding the UBQ-2 protein from the <i>T. canis</i>-omic dataset, called Tcubq-2. After cloning and sequencing, we conducted sequence analysis and structural modeling of TcUBQ-2 using online bioinformatics tools. The recombinant TcUBQ-2 protein (rTcUBQ-2) was prokaryotically expressed and subjected to Western blot analysis to assess its antigenicity and immunoreactivity. Additionally, we performed immunolocalization of the endogenous protein in adult <i>T. canis</i> and evaluated its serodiagnostic potential using sera from naïve and infected mice and dogs. Our results showed that the complete cDNA sequence of Tcubq-2 was 387 bp in length and encoded a polypeptide of 128 amino acids, lacking both an N-terminal signal sequence and a transmembrane domain. Sequence and phylogenetic analyses showed that TcUBQ-2 shared the closest genetic distance with its homologs in <i>Parascaris univalens</i> and <i>Ascaris suum</i>. Real-time quantitative PCR and Western blotting revealed an expression peak of TcUBQ-2 in the intestine-hatched second-stage (L3) larvae compared to other developmental stages of <i>T. canis</i>. Tissue localization of endogenous TcUBQ-2 revealed its broad distributions in the body wall, muscle, gut epithelium, and microvilli of adult <i>T. canis,</i> with gender-specific expression in the uterus, ovary, and non-embryonated eggs of females. Based on its strong immunogenic properties, a recombinant TcUBQ-2 (rTcUBQ-2)-based ELISA was established and exhibited a sensitivity of 100% and a specificity of 95.8% to detect anti-<i>T. canis</i> mouse sera and a sensitivity of 79.2% and a specificity of 83.3% to detect anti-<i>T. canis</i> dog sera, respectively. This study presents a comprehensive bioinformatics analysis of the dog roundworm TcUBQ-2, and its strong serodiagnostic performance suggests that rTcUBQ-2 has the potential to be developed into an ELISA-based serological test for detecting toxocariasis in dogs and other accidental hosts, including humans.