Fujian Provincial Key Laboratory of Developmental Biology and Neuroscience, College of Life Science, Fujian Normal University, Fuzhou, Fujian, China.
Parasitology. 2013 Apr;140(5):561-8. doi: 10.1017/S0031182012001977. Epub 2013 Jan 4.
Actin depolymerizing factors regulate actin dynamics involved in cellular processes such as morphogenesis, motility, development and infection. Here, a novel actin depolymerizing factor gene (CiADF 2 ) was cloned from the cDNA library of Cryptocaryon irritans, a parasitic ciliate causing cryptocaryonosis. The full-length cDNA of CiADF 2 was 531 bp. Its open reading frame (ORF) was 417 bp, encoding a polypeptide of 138 aa with typical features of the ADF/cofilin family. Reverse transcription-PCR suggested that CiADF 2 is expressed in all stages of the life cycle. After site-directed mutagenesis of a non-universal genetic code, the ORF was subcloned in Escherichia coli. The bacteria were induced with the addition of isopropylthio-β-D-galactoside to express a fusion protein of recombinant CiADF2 (rCiADF2) with glutathione S transferase. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot confirmed the predicted molecular mass of rCiADF2 of 16·2 kDa. A mouse antibody against rCiADF2 recognized native CiADF2, and rCiADF2 reacted with mouse antisera against C. irritans trophonts. CiADF2 was abundant in the plasma around cytostomes, suggesting that CiADF2 is involved in ciliate movement. Moreover, rCiADF2 showed F-actin binding and depolymerizing activity. This study will help to clarify the pathogenic biology of the parasite and develop effective control measures for cryptocaryonosis.
肌动蛋白解聚因子调节细胞过程中的肌动蛋白动力学，如形态发生、运动、发育和感染。在这里，从 Cryptocaryon irritans 的 cDNA 文库中克隆了一种新型肌动蛋白解聚因子基因（CiADF 2 ），Cryptocaryon irritans 是一种引起隐鞭虫病的寄生纤毛虫。CiADF 2 的全长 cDNA 为 531bp。其开放阅读框（ORF）为 417bp，编码一个 138aa 的多肽，具有 ADF/cofilin 家族的典型特征。逆转录 PCR 表明 CiADF 2 在生命周期的所有阶段都有表达。在对非通用遗传密码进行定点突变后，将 ORF 亚克隆到大肠杆菌中。通过添加异丙基硫代-β-D-半乳糖苷诱导细菌表达与谷胱甘肽 S 转移酶融合的重组 CiADF2（rCiADF2）融合蛋白。十二烷基硫酸钠聚丙烯酰胺凝胶电泳和 Western blot 证实 rCiADF2 的预测分子质量为 16.2kDa。针对 rCiADF2 的小鼠抗体识别天然 CiADF2，并且 rCiADF2 与针对 C. irritans 营养体的小鼠抗血清反应。CiADF2 在胞口周围的血浆中含量丰富，表明 CiADF2 参与纤毛虫运动。此外，rCiADF2 显示出 F-肌动蛋白结合和解聚活性。这项研究将有助于阐明寄生虫的致病生物学，并开发针对隐鞭虫病的有效控制措施。
