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Eukaryotic elongation factor 2 is involved in the anticoccidial action of diclazuril in the second-generation merozoites of Eimeria tenella.
Veterinary Parasitology ( IF 2.6 ) Pub Date : 2019-11-17 , DOI: 10.1016/j.vetpar.2019.108991 Bian-Hua Zhou 1 , Liu-Shu Jia 1 , Hong-Wei Guo 2 , Hai-Yan Ding 1 , Jing-Yun Yang 1 , Hong-Wei Wang 1
Veterinary Parasitology ( IF 2.6 ) Pub Date : 2019-11-17 , DOI: 10.1016/j.vetpar.2019.108991 Bian-Hua Zhou 1 , Liu-Shu Jia 1 , Hong-Wei Guo 2 , Hai-Yan Ding 1 , Jing-Yun Yang 1 , Hong-Wei Wang 1
Affiliation
Eimeria tenella, an obligate intracellular parasite, can actively invade the cecal epithelial cells of chickens and cause severe enteric disease. Eukaryotic elongation factor 2 (eEF2) plays a major role in protein synthesis and cell survival. This study aims to explore the exact mechanisms underlying diclazuril inhibition in second-generation merozoites of E. tenella. The eEF2 cDNA of the second-generation merozoites of E. tenella (EtEF2) was cloned by reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Diclazuril-induced expression profiles of EtEF2 were also analyzed. The cloned full-length cDNA (2893 bp) of the EtEF2 nucleotide sequence encompassed a 2499 bp open reading frame (ORF) that encoded a polypeptide of 832 residues with an estimated molecular mass of 93.12 kDa and a theoretical isoelectric point of 5.99. The EtEF2 nucleotide sequence was submitted to the GenBank database with the accession number KF188423. The EtEF2 protein sequence shared 99 % homology with the eEF2 sequence of Toxoplasma gondii (GenBank XP_002367778.1). The GTPase activity domain and ADP-ribosylation domain were conserved signature sequences of the eEF2 gene family. The changes in the transcriptional and translational levels of EtEF2 were detected through quantitative real-time PCR and Western blot analyses. The mRNA expression level of EtEF2 was 2.706 fold increases and the protein level of EtEF2 was increased 67.31 % under diclazuril treatment. In addition, the localization of EtEF2 was investigated through immunofluorescence assay. Experimental results demonstrated that EtEF2 was distributed primarily in the cytoplasm of second-generation merozoites, and its fluorescence intensity was enhanced after diclazuril treatment. These findings indicated that EtEF2 may have an important role in understanding the signaling mechanism underlying the anticoccidial action of diclazuril and could be a promising target for novel drug exploration.
中文翻译:
真核生物伸长因子2参与地克珠利在艾美球虫第二代裂殖子中的抗球虫作用。
艾美耳球虫是一种专性的细胞内寄生虫,可主动侵入鸡的盲肠上皮细胞,并引起严重的肠病。真核延伸因子2(eEF2)在蛋白质合成和细胞存活中起主要作用。这项研究旨在探讨确切的机制,抑制地克珠菌抑制第二代裂殖芽孢杆菌的裂殖子。通过逆转录酶聚合酶链反应和快速扩增cDNA末端,克隆了第二代棉球虫(EtEF2)裂殖子的eEF2 cDNA。还分析了地克珠利诱导的EtEF2表达谱。克隆的EtEF2核苷酸序列的全长cDNA(2893 bp)包含2499 bp的开放阅读框(ORF),其编码832个残基的多肽,估计分子量为93.12 kDa,理论等电点为5。99. EtEF2核苷酸序列已提交至GenBank数据库,登录号为KF188423。EtEF2蛋白序列与弓形体(GenBank XP_002367778.1)的eEF2序列具有99%的同源性。GTPase活性域和ADP-核糖基化域是eEF2基因家族的保守签名序列。EtEF2的转录和翻译水平的变化通过实时定量PCR和Western印迹分析进行检测。在地克珠利处理下,EtEF2的mRNA表达水平增加了2.706倍,EtEF2的蛋白水平增加了67.31%。另外,通过免疫荧光测定法研究了EtEF2的定位。实验结果表明,EtEF2主要分布在第二代裂殖子的细胞质中,地克珠利处理后荧光强度增强。这些发现表明,EtEF2在理解地克珠利抗球虫作用的信号传导机制中可能起重要作用,并且可能成为新药探索的有希望的靶标。
更新日期:2019-11-01
中文翻译:
真核生物伸长因子2参与地克珠利在艾美球虫第二代裂殖子中的抗球虫作用。
艾美耳球虫是一种专性的细胞内寄生虫,可主动侵入鸡的盲肠上皮细胞,并引起严重的肠病。真核延伸因子2(eEF2)在蛋白质合成和细胞存活中起主要作用。这项研究旨在探讨确切的机制,抑制地克珠菌抑制第二代裂殖芽孢杆菌的裂殖子。通过逆转录酶聚合酶链反应和快速扩增cDNA末端,克隆了第二代棉球虫(EtEF2)裂殖子的eEF2 cDNA。还分析了地克珠利诱导的EtEF2表达谱。克隆的EtEF2核苷酸序列的全长cDNA(2893 bp)包含2499 bp的开放阅读框(ORF),其编码832个残基的多肽,估计分子量为93.12 kDa,理论等电点为5。99. EtEF2核苷酸序列已提交至GenBank数据库,登录号为KF188423。EtEF2蛋白序列与弓形体(GenBank XP_002367778.1)的eEF2序列具有99%的同源性。GTPase活性域和ADP-核糖基化域是eEF2基因家族的保守签名序列。EtEF2的转录和翻译水平的变化通过实时定量PCR和Western印迹分析进行检测。在地克珠利处理下,EtEF2的mRNA表达水平增加了2.706倍,EtEF2的蛋白水平增加了67.31%。另外,通过免疫荧光测定法研究了EtEF2的定位。实验结果表明,EtEF2主要分布在第二代裂殖子的细胞质中,地克珠利处理后荧光强度增强。这些发现表明,EtEF2在理解地克珠利抗球虫作用的信号传导机制中可能起重要作用,并且可能成为新药探索的有希望的靶标。