Chen Lin, Yang Deying, Gu Xiaobin, Peng Xuerong, Yang Guangyou
Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Ya'an 625014, China.
College of Life and Basic Science, Sichuan Agricultural University, Ya'an 625014, China.
Vet Parasitol. 2014 Aug 29;204(3-4):214-20. doi: 10.1016/j.vetpar.2014.05.021. Epub 2014 May 24.
Cysticercosis, caused by the larvae of Taenia pisiformis, is a common disease in domestic breeds of the rabbit Oryctolagus cuniculus that results in economic losses. At present, there is no convenient and effective method for the rapid detection of T. pisiformis larvae. Here, we developed and tested the efficacy of a Dot-ELISA assay for the diagnosis of T. pisiformis larval infections in rabbits, based on the expression of the recombinant fusion protein (rTp1) from the Tp1 gene. Rapid amplification of cDNA ends (RACE) was used to amplify the 3' ends of the Tp1 gene, based on the unigene similar to Ts1 gene (EU009656.1) which comes from transcriptome sequencing of T. pisiformis. The Tp1 gene was successfully amplified, cloned and expressed in BL21 (DE3). Western blot analysis revealed that the recombinant Tp1 protein is specifically recognized by rabbit T. pisiformis cysticercosis antisera. This purified recombinant fusion protein, rTp1, was probed by Dot-ELISA with sera from rabbits infected with T. pisiformis larvae and with other parasitic infections. Results showed that this Dot-ELISA assay had both high sensitivity (92.9-97.6%) and specificity (95.2-98.4%) to detect T. pisiformis larval infections. We also found very low levels of cross-reaction with other parasitic infections. This study has revealed that our novel Dot-ELISA assay utilizing the recombinant fusion protein, rTp1, has a strong potential for the effective diagnosis of T. pisiformis infections in rabbits.
由豆状带绦虫幼虫引起的囊尾蚴病是家兔品种穴兔的一种常见疾病，会造成经济损失。目前，尚无便捷有效的方法来快速检测豆状带绦虫幼虫。在此，我们基于来自Tp1基因的重组融合蛋白（rTp1）的表达，开发并测试了一种用于诊断兔豆状带绦虫幼虫感染的斑点酶联免疫吸附测定（Dot-ELISA）方法的有效性。基于来自豆状带绦虫转录组测序的与Ts1基因（EU009656.1）相似的单基因，采用cDNA末端快速扩增（RACE）技术扩增Tp1基因的3'末端。Tp1基因在BL21（DE3）中成功扩增、克隆并表达。蛋白质免疫印迹分析表明，重组Tp1蛋白能被兔豆状带绦虫囊尾蚴病抗血清特异性识别。用感染豆状带绦虫幼虫的兔血清和其他寄生虫感染的血清通过斑点酶联免疫吸附测定法检测这种纯化的重组融合蛋白rTp1。结果表明，这种斑点酶联免疫吸附测定法检测豆状带绦虫幼虫感染具有高灵敏度（92.9 - 97.6%）和高特异性（95.2 - 98.4%）。我们还发现与其他寄生虫感染的交叉反应水平非常低。这项研究表明，我们利用重组融合蛋白rTp1的新型斑点酶联免疫吸附测定法在有效诊断兔豆状带绦虫感染方面具有强大潜力。
