Design and expression of polytopic construct of cathepsin-L1, SAP-2 and FhTP16.5 proteins of Fasciola hepatica

Abstract

The enzyme-linked immunosorbent assay (ELISA) technique can play an important role in the early detection of fascioliasis. However, they have some diagnostic limitations, including cross-reaction with other helminths. It seems that the combination of recombinant parasite proteins as antigen can reduce these problems. Hence, the present study was aimed to design and confirm the antigenic recombinant multi-epitope (rMEP) construct of three protein epitopes (linear and conformational B-cell epitopes) of the parasite using immunoinformatic tools. For this purpose, the tertiary structures of Fasciola hepatica cathepsin-L1, saposin-like protein 2 and 16.5-kDa tegument-associated protein were predicted using the I-TASSER server. Validation of the modelled structures was performed by Ramachandran plots. The antigenic epitopes of the proteins were achieved by analysing the features of the IEDB server. The synthesized gene was cloned into the pET-22b (+) expression vector and transformed into the Escherichia coli BL21. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to verify and analyse the expression of the rMEP protein. Western blotting was utilized to confirm rMEP protein immunogenicity in two forms, one using an anti-His tag antibody and the other with human pooled sera samples (fascioliasis, non-fascioliasis and negative control sera). Our results demonstrated that the rMEP designed for the three proteins of F. hepatica was highly antigenic, and immune-detection techniques confirmed the antigen specificity. In conclusion, the presented antigenic multi-epitope may be very helpful to develop serodiagnostic kits such as indirect ELISA to evaluate the proper diagnosis of fascioliasis in humans and ruminants.

High relatedness of bioinformatic data and realistic experimental works on the potentials of Fasciola hepatica and F. gigantica cathepsin L1 as a diagnostic and vaccine antigen

“…Even for human sera, FhCatL1 and FgCatL1 have demonstrated an excellent ability in detecting specific antibody in infected patients with very low cross-reactivity. Many studies have utilized the indirect ELISAs based on rFhCatL1 ( 19 , 54 – 60 ). Consistently, FgCatL1 based ELISAs showed an optimal performance against human sera when used as a recombinant protein ( 61 , 62 ), or specific peptides prepared from rFgCatL1 ( 63 , 64 ).…”

“…Few studies have analyzed the potentials of FhCatL1 as diagnostic or vaccine antigens using bioinformatic tools and much less for FgCatL1 ( 19 , 20 ). Nevertheless, such studies have only focused on certain properties and using few analysis tools.…”

Bioinformatics analysis for the purpose of designing a novel multi-epitope DNA vaccine against Leishmania major

“…Peptide vaccines are potentially a new technology to struggle with the Leishmania infection. Bioinformatics procedures for the analysis of proteins have become an essential instrument for vaccine researches 13,17,18 . The prediction of epitopes is a significant facility for designing new vaccines 4 .Nowadays use of immune-informatics software, have led to save a great deal of time to design vaccine constructs.…”

Challenges in Serologic Diagnostics of Neglected Human Systemic Mycoses: An Overview on Characterization of New Targets

“…There are several successful examples of the application of immunoinformatics in the identification/development of new diagnostic targets as described with COVID-19 [ 185 , 186 , 187 ], Schistosoma mansoni [ 188 ], Toxoplasma gondii [ 189 ] Fasciola hepatica [ 190 ], helminth [ 191 ] human brucellosis [ 192 ] and Leishmania [ 193 ]. In the context of systemic mycosis, the number of works is substantially limited, and this approach has been used combined with immunoproteomic analyses as described to Paracoccidioides complex [ 43 , 172 ], Sporothrix schenckii complex [ 159 ] and Cryptococcus gattii [ 158 ].…”