Key Laboratory of Veterinary Drug Safety Evaluation and Residues Research, Chinese Academy of Agricultural Sciences, Shanghai Veterinary Research Institute, 518 Ziyue Road, Minhang, Shanghai, 200241, People's Republic of China.
Parasitol Res. 2010 Feb;106(3):571-6. doi: 10.1007/s00436-009-1699-z. Epub 2009 Dec 18.
Actin depolymerizing factor (ADF) is an essential actin-binding protein that plays a key role in the control of actin dynamics and actin-based motility processes in intracellular parasites. To determine the effects of diclazuril on ADF gene of second-generation merozoites (mz-ADF) mRNA expression in Eimeria tenella, mz-ADF gene was cloned by RT-PCR from extracted RNA in second-generation merozoite of E. tenella and successfully expressed by pET-28a vector in Escherichia coli BL21(DE3). Results showed that the full length of the cloned cDNA sequence of the mz-ADF gene is 476 bp including an ORF of 375 bp. The sequence has 100% homology with a published sequence of sporozoite stage E. tenella ADF mRNA (GenBank EF195234.1). The recombinant protein was induced to be expressed by 1 mM isopropyl beta-D: -1-thiogalactopyranoside in vitro. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that 16.99 kDa fusion protein existed in solvable form. Compared with the infected/control group, mz-ADF mRNA expression level was downregulated by 63.86% in the infected/treatment group with the treatment of diclazuril. In conclusion, the data presented here indicate that mz-ADF gene participates in an important role in the invasion host of E. tenella. Downregulation of mz-ADF mRNA expression enrich the mechanism study of diclazuril on E. tenella.
肌动蛋白解聚因子(ADF)是一种重要的肌动蛋白结合蛋白,在控制细胞内寄生虫的肌动蛋白动力学和基于肌动蛋白的运动过程中起着关键作用。为了确定双氯苯双胍对柔嫩艾美耳球虫第二代裂殖子(mz-ADF)mRNA 表达的影响,本研究通过 RT-PCR 从柔嫩艾美耳球虫第二代裂殖子中提取的 RNA 克隆 mz-ADF 基因,并成功在大肠杆菌 BL21(DE3)中通过 pET-28a 载体表达。结果表明,克隆的 mz-ADF 基因 cDNA 序列全长为 476bp,包含一个 375bp 的 ORF。该序列与已发表的柔嫩艾美耳球虫裂殖子阶段 ADF mRNA(GenBank EF195234.1)的序列具有 100%的同源性。重组蛋白在体外通过 1mM 异丙基-β-D:-1-硫代半乳糖吡喃糖苷诱导表达。SDS-PAGE 分析表明,可溶形式存在 16.99kDa 的融合蛋白。与感染/对照组相比,双氯苯双胍处理组的 mz-ADF mRNA 表达水平下调了 63.86%。综上所述,本研究结果表明 mz-ADF 基因参与了柔嫩艾美耳球虫入侵宿主的过程。mz-ADF mRNA 表达下调丰富了双氯苯双胍对柔嫩艾美耳球虫作用机制的研究。