University of Firat, Faculty of Veterinary Medicine, Department of Parasitology, Elazig, Turkey.
University of Firat, Faculty of Veterinary Medicine, Department of Parasitology, Elazig, Turkey.
Vet Parasitol. 2023 Aug;320:109961. doi: 10.1016/j.vetpar.2023.109961. Epub 2023 May 27.
Fasciolosis caused by Fasciola hepatica is a disease of zoonotic importance that is common worldwide and can cause serious problems in farm animals, some wild animals and humans. The development of diagnostic kits for the correct diagnosis of fasciolosis in sheep is important in terms of preventing yield losses. With this study, it is aimed to clone and express the enolase gene to be isolated from adult F. hepatica and to determine the effectiveness of the recombinant antigen in the serodiagnosis of sheep fasciolosis. For this aim, primers were designed to amplify the enolase gene from the F. hepatica enolase sequence, mRNA was isolated from F. hepatica adult fluke obtained from an infected sheep followed by cDNA was obtained. Enolase gene was amplified by PCR and the product was cloned and then expressed. The efficiency of the purified recombinant protein was displayed by Western blot (WB) and ELISA using positive and negative sheep sera. As a result, the sensitivity and specificity rates of the recombinant FhENO antigen were 85% and %82.8 by WB while the rates were 90% and 97.14% by ELISA, respectively. At the same time, in sheep blood sera samples collected from the Elazig and Siirt provinces of Turkey, 100 (50%) of 200 sera were found to be positive by WB and 46 (23%) were found to be positive by ELISA. The most important problem in ELISA was the high cross-reaction rate of the recombinant antigen used, as in WB. In order to prevent the cross-reactions, it will be useful to compare the genes encoding the enolase protein of parasites from the closely related parasite family, and select the regions where there are no common epitopes, and clone them and test the purified protein.
由肝片吸虫引起的片形吸虫病是一种具有动物传染病重要性的疾病，在世界范围内很常见，可给农场动物、一些野生动物和人类造成严重问题。开发用于正确诊断绵羊片形吸虫病的诊断试剂盒对于防止产量损失很重要。本研究旨在从成年肝片吸虫中克隆和表达分离出的烯醇酶基因，并确定重组抗原在绵羊片形吸虫病血清学诊断中的有效性。为此，设计了从 F. hepatica 烯醇酶序列中扩增烯醇酶基因的引物，从感染绵羊获得的成年肝片吸虫中分离 mRNA，然后获得 cDNA。通过 PCR 扩增烯醇酶基因，将产物克隆并表达。使用阳性和阴性绵羊血清通过 Western blot（WB）和酶联免疫吸附试验（ELISA）显示纯化重组蛋白的效率。结果，重组 FhENO 抗原的 WB 敏感性和特异性分别为 85%和 82.8%，ELISA 分别为 90%和 97.14%。同时，在土耳其埃拉泽和锡尔特省采集的绵羊血液血清样本中，WB 检测到 200 份血清中有 100 份（50%）为阳性，ELISA 检测到 46 份（23%）为阳性。在 ELISA 中最重要的问题是重组抗原的高交叉反应率，与 WB 一样。为了防止交叉反应，比较来自密切相关寄生虫家族的寄生虫烯醇酶蛋白编码基因，并选择没有共同表位的区域进行克隆和测试纯化蛋白将很有用。
