Expression, purification and in vitro refolding of the recombinant truncated Saposin-like protein 2 antigen for development of diagnosis of human fascioliasis

- rtSAP2 expressed in Shuffle® T7 and Rosetta strains in soluble and insoluble forms.
- rtSAP-2 was purified under native and denatured conditions.
- Accurate folding of rtSAP2 was evaluated in western blots and ELISA.
- Refolded rtSAP2 by dialysis method detected all of fasciola-infected samples.
- Refolded protein is more appropriate than unfolded and FhE/S forms for diagnosis.

Early diagnosis of fascioliasis is critical in prevention of injury to the liver and bile ducts. Saposin-like protein (FhSAP-2) is probably the most ideal antigen of Fasciola hepatica for development of ELISA kits. SAP-2 has a conserved tertiary structure containing three disulfide bonds and conformational epitopes. Therefore, antigenicity of SAP-2 is greatly depends on disulfide bond formation and proper folding. We produced the recombinant truncated SAP-2 (rtSAP-2) in the SHuffle® T7 and Rosetta strain of Escherichia coli, in soluble and insoluble forms, respectively and purified by immobilized metal affinity chromatography (IMAC). The refolding process of denatured rtSAP-2 was performed using dialysis and dilution methods in the presence of chemical additives, along with reduced/oxidized glutathione (in vitro). Physicochemical studies, including non-reducing gel electrophoresis, Ellman's assay, Western blotting and ELISA showed the most antigenicity and likely correct folding of rtSAP-2, which was obtained by dialysis method. An IgG ELISA test was developed using rtSAP-2 refolded by dialysis and compared with excretory/secretory products of parasite with 52 positive fascioliasis samples, 79 other parasitic samples and 70 negative controls samples. The results exhibited 100% sensitivity and 98% specificity for rtSAP-2, also, 100% and 95.3% for excretory/secretory (E/S) antigen, respectively. In conclusion, it is suggested that rtSAP-2 with the correct folding could be used as a candidate antigen for detection of human fascioliasis.

184Evaluation of antigenicity of refolded rtSAP-2 protein by in vitro method in compared with E/S antigens for diagnosis of human fascioliasis.