Zhu Pengchen, Hu Xin, Huang Jiali, Wang Lidan, Wang Zhao, Wang Ruixi, Fan Xinyi, Wei Guoshan, He Qin, Li Zhaoruiyi, Zhou Xuan, Wang Hui, Xie Yue
Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.
Beijing Key Laboratory of Captive Wildlife Technologies, Beijing Zoo, Beijing, China.
Front Vet Sci. 2025 Mar 31;12:1550489. doi: 10.3389/fvets.2025.1550489. eCollection 2025.
Toxocariasis, caused by the dog roundworm 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 -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 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 and . 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 . Tissue localization of endogenous TcUBQ-2 revealed its broad distributions in the body wall, muscle, gut epithelium, and microvilli of adult 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- mouse sera and a sensitivity of 79.2% and a specificity of 83.3% to detect anti- 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.
犬弓首蛔虫引起的弓首蛔虫病是一种全球分布的人畜共患寄生虫病，对兽医和公共卫生构成重大威胁。寄生虫中的泛素加一种L40核糖体蛋白（UBQ-2）在蛋白质降解和减数分裂中起关键作用，从而影响寄生虫的发育、存活以及寄生虫与宿主的相互作用。在本研究中，我们首次从组学数据集中鉴定出一个编码UBQ-2蛋白的全长cDNA，称为Tcubq-2。经过克隆和测序后，我们使用在线生物信息学工具对TcUBQ-2进行了序列分析和结构建模。重组TcUBQ-2蛋白（rTcUBQ-2）通过原核表达，并进行蛋白质免疫印迹分析以评估其抗原性和免疫反应性。此外，我们对成虫体内的内源性蛋白进行了免疫定位，并使用未感染和感染的小鼠及犬的血清评估了其血清学诊断潜力。我们的结果表明，Tcubq-2的完整cDNA序列长度为387 bp，编码一个128个氨基酸的多肽，既没有N端信号序列也没有跨膜结构域。序列和系统发育分析表明，TcUBQ-2与其在[具体物种1]和[具体物种2]中的同源物具有最接近的遗传距离。实时定量PCR和蛋白质免疫印迹显示，与犬弓首蛔虫的其他发育阶段相比，TcUBQ-2在肠道孵化的第二期（L3）幼虫中表达量达到峰值。内源性TcUBQ-2的组织定位显示其在成虫的体壁、肌肉、肠道上皮和微绒毛中广泛分布，在雌性的子宫、卵巢和未受精卵中有性别特异性表达。基于其强大的免疫原性，建立了一种基于重组TcUBQ-2（rTcUBQ-2）的ELISA，检测抗小鼠血清时灵敏度为100%，特异性为95.8%，检测抗犬血清时灵敏度为79.2%，特异性为83.3%。本研究对犬弓首蛔虫TcUBQ-2进行了全面的生物信息学分析，其强大的血清学诊断性能表明rTcUBQ-2有潜力被开发成一种基于ELISA的血清学检测方法，用于检测犬及其他偶然宿主（包括人类）中的弓首蛔虫病。
