title: Receptor for activated C kinase ortholog of second-generation merozoite in Eimeria tenella: clone, characterization, and diclazuril-induced mRNA expression.
abstract: The receptor for activated C kinase (RACK) cDNA of second-generation merozoites of Eimeria tenella was cloned using reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends, compared with other species, and then successfully expressed using the pET-28a vector in Escherichia coli BL21 (DE3) (EtRACK). Nucleotide sequence analysis revealed that the full length of the cloned cDNA (1,264 bp) encompassed a 957-bp open reading frame encoding a polypeptide of 318 residues with an estimated molecular mass of 34.94 kDa and a theoretical isoelectric point of 5.97. Molecular analysis of EtRACK reveals the presence of seven WD40 repeat motifs. EtRACK localizes to the cytoplasm and nucleus in second-generation merozoites of E. tenella. The cDNA sequence has been submitted to the GenBank Database with accession number JQ292804. EtRACK shared 98% homology with the published sequence of a RACK protein from Toxoplasma gondii at the amino acid level (GenBank XP_002370996.1). Recombinant protein expression was induced using 1 mM of isopropyl β-D-1-thiogalactopyranoside in vitro at 30 °C. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that the 39.79-kDa fusion protein existed in unsolvable form. Quantitative real-time PCR analysis showed that compared with the control group, the level of EtRACK mRNA expression in the treatment group was downregulated by 81.3% by diclazuril treatment. The high similarity of EtRACK to previously described RACKs of other organisms, as well as its downregulated expression in second-generation merozoites induced by diclazuril, suggests that it could play a key role in the signaling event that precedes protein secretion and parasite invasion. Moreover, the downregulation of EtRACK mRNA expression also enriches studies on the mechanism of action of diclazuril on E. tenella.
authors: Key Laboratory of Veterinary Drug Safety Evaluation and Residues Research, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 518 Ziyue Road, Minhang, Shanghai 200241, People's Republic of China.
authors: Bian-hua
authors: BH
authors: Zhou
authors: Xiao-jiong
authors: XJ
authors: Shen
authors: Hong-wei
authors: HW
authors: Wang
authors: Tao
authors: T
authors: Li
authors: Fei-qun
authors: FQ
authors: Xue
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D016786:Eimeria tenella / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D020869:Gene Expression Profiling; D007526:Isoelectric Point; D052940:Merozoites; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009570:Nitriles / Q000378:metabolism*; D016366:Open Reading Frames; D012333:RNA, Messenger / Q000096:biosynthesis*; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014122:Toxoplasma / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects*; D014227:Triazines / Q000378:metabolism*
chemical_list: D016054:DNA, Protozoan; D009570:Nitriles; D012333:RNA, Messenger; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D014227:Triazines; C057884:diclazuril