title: Actin-depolymerizing factor of second-generation merozoite in Eimeria tenella: clone, prokaryotic expression, and diclazuril-induced mRNA expression.
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 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.
authors.affiliation: 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. zbh210@163.com
authors.forename: Bian-hua
authors.initials: BH
authors.lastname: Zhou
authors.forename: Hong-wei
authors.initials: HW
authors.lastname: Wang
authors.forename: Fei-qun
authors.initials: FQ
authors.lastname: Xue
authors.forename: Xiao-yang
authors.initials: XY
authors.lastname: Wang
authors.forename: Feng-kun
authors.initials: FK
authors.lastname: Yang
authors.forename: Man-man
authors.initials: MM
authors.lastname: Ban
authors.forename: Rui-xiang
authors.initials: RX
authors.lastname: Xin
authors.forename: Cong-cong
authors.initials: CC
authors.lastname: Wang
mesh_terms: D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D001483:Base Sequence; D002645:Chickens / Q000469:parasitology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D051345:Destrin / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D016786:Eimeria tenella / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008297:Male; D052940:Merozoites / Q000201:enzymology*; D008969:Molecular Sequence Data; D009570:Nitriles / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D014227:Triazines / Q000494:pharmacology*
chemical_list: D000981:Antiprotozoal Agents; D018076:DNA, Complementary; D051345:Destrin; D009570:Nitriles; D012333:RNA, Messenger; D014227:Triazines; C057884:diclazuril