PubAg

Main content area

Actin-depolymerizing factor of second-generation merozoite in Eimeria tenella: clone, prokaryotic expression, and diclazuril-induced mRNA expression

Author:
Zhou, Bian-hua, Wang, Hong-wei, Xue, Fei-qun, Wang, Xiao-yang, Yang, Feng-kun, Ban, Man-man, Xin, Rui-xiang, Wang, Cong-cong
Source:
Parasitology research 2010 v.106 no.3 pp. 571-576
ISSN:
0932-0113
Subject:
Eimeria tenella, Escherichia coli, actin, complementary DNA, diclazuril, gene expression, genes, merozoites, messenger RNA, open reading frames, parasites, polyacrylamide gel electrophoresis, reverse transcriptase polymerase chain reaction
Abstract:
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 β-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.
Agid:
638724