National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan; Department of Biochemistry and Chemistry of Nutrition, Faculty of Veterinary Medicine, Sadat City University, Sadat City, Menoufyia, Egypt.
Exp Parasitol. 2013 Oct;135(2):414-20. doi: 10.1016/j.exppara.2013.08.005. Epub 2013 Aug 19.
Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs.
鉴定和分子特征 Babesia gibsoni 蛋白具有潜在的抗原性，对于开发和验证血清学诊断方法至关重要。在本研究中，我们通过免疫筛选寄生虫 cDNA 文库分离到一个编码 B. gibsoni 76-kDa 蛋白的 cDNA 克隆。计算机分析表明，该蛋白在 C 末端具有富含谷氨酸的区域。因此，该蛋白被命名为 B. gibsoni 谷氨酸丰富蛋白（BgGARP）。对 BgGARP 多肽的 BLASTp 分析表明，该肽与 Babesia bigemina 和 Babesia bovis 的 200-kDa 蛋白具有显著同源性。一个编码预测 13-kDa 肽的截断 BgGARP cDNA（BgGARPt）在大肠杆菌（E. coli）中表达，并用针对重组蛋白的鼠抗血清来鉴定相应的天然蛋白。针对重组 BgGARPt（rBgGARPt）的抗血清识别感染红细胞裂解物中的 140-kDa 蛋白，通过共聚焦显微镜观察可在寄生虫的细胞质中检测到该蛋白。此外，使用 rBgGARPt 评估了酶联免疫吸附试验（ELISA）的特异性和敏感性，方法是使用 B. gibsoni 感染犬血清和特定病原体无（SPF）犬血清。此外，使用 rBgGARPt ELISA 检测了临床上诊断为巴贝斯虫病的 107 份犬血清样本。结果表明，rBgGARPt-ELISA 阳性的样本有 86（80.4%）份，与 IFAT 和 PCR 作为参考试验相当。总之，这些结果表明 BgGARP 是一种适合检测犬抗 B. gibsoni 抗体的血清学诊断抗原。
