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.
Parasitol Res. 2012 Oct;111(4):1447-55. doi: 10.1007/s00436-012-2978-7. Epub 2012 Jun 30.
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.
柔嫩艾美耳球虫第二代裂殖子激活蛋白激酶受体（RACK）cDNA 的克隆及其在大肠杆菌中的表达
采用逆转录聚合酶链反应（RT-PCR）和快速扩增 cDNA 末端技术（RACE），从柔嫩艾美耳球虫第二代裂殖子中克隆了 RACK cDNA，与其他物种相比，成功地在大肠杆菌 BL21（DE3）（EtRACK）中利用 pET-28a 载体进行了表达。核苷酸序列分析表明，克隆 cDNA 的全长（1264bp）包含一个 957bp 的开放阅读框，编码一个 318 个氨基酸的多肽，预计分子量为 34.94kDa，理论等电点为 5.97。EtRACK 的分子分析表明，它存在七个 WD40 重复基序。EtRACK 定位于柔嫩艾美耳球虫第二代裂殖子的细胞质和细胞核中。该 cDNA 序列已被提交到 GenBank 数据库，登录号为 JQ292804。EtRACK 在氨基酸水平上与已发表的刚地弓形虫 RACK 蛋白序列（GenBank XP_002370996.1）同源性为 98%。在 30°C 下，用 1mM 的异丙基-β-D-1-硫代半乳糖吡喃糖苷（IPTG）体外诱导重组蛋白表达。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表明，39.79kDa 的融合蛋白以不可溶的形式存在。实时定量 PCR 分析表明，与对照组相比，二氯苯脲处理组 EtRACKmRNA 表达水平下调 81.3%。EtRACK 与其他生物中已描述的 RACK 具有高度相似性，并且二氯苯脲诱导第二代裂殖子中 EtRACK 的表达下调，表明它可能在蛋白分泌和寄生虫入侵之前的信号事件中发挥关键作用。此外，EtRACKmRNA 表达下调也丰富了二氯苯脲对柔嫩艾美耳球虫作用机制的研究。
