Cloning and expression of the fasciola hepatica enolase gene and determination of the efficacy of recombinant protein in the diagnosis of sheep fasciolosis

Fasciolosis, a neglected disease, is an infection of the liver caused by two main pathogenic species, Fasciola hepatica and Fasciola gigantica, and other digenetic trematodes in the Fasciolidae family. Fasciolosis caused by F. hepatica is a disease of zoonotic importance that is common worldwide and can cause serious problems in both humans and livestocks. The development of diagnostic kits for the proper diagnosis of fasciolosis in sheep is highly important in terms of preventing yield losses. Studies with the enzyme enolase, one of the Excrete/Secrete (E/S) products of F. hepatica, have shown that this enzyme may be an important protective component in the tegument of the fluke against sheep fasciolosis. In this thesis, it was aimed to clone and express the enolase gene isolated from the adult F. hepatica fluke obtained from sheep and to determine the effectiveness of the recombinant antigen in the diagnosis of sheep fasciolosis. For this aim, primers were designed from the enolase gene fragment sequences of F. hepatica. Subsequently, mRNA was isolated from adult F. hepatica and cDNA was obtained. The gene region related to the designed primers was amplified by PCR and the product obtained was cloned and the protein was expressed. The activity of the purified recombinant protein was then determined by Western blot (WB) and ELISA using positive and negative sheep sera. As a result, the sensitivity of recombinant FhENO was 85% with WB and 90% with ELISA while the specificity was determined as 82.8% by WB and 97.14% by ELISA. At the same time, in sheep sera samples collected from Elazig and Siirt provinces of Turkey, 100 out of 200 (50%) sera were found to be positive by WB and 46 (23%) were found to be positive by ELISA. It has been shown that recombinant FhENO antigen can be used in a WB and ELISA-based test in the diagnosis of fasciolosis in sheep. However, 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.