LAUSR

Toxoplasma gondii is an intracellular protozoan that infects a wide range of mammalians. Commercial diagnostic kits usually use the whole parasite antigen to, which can reduce the specificity of the test. B-cell epitopes are antigenic structural index and react specifically with antibody receptors. Prediction of B-cell epitopes and designing of antigenic multi-epitope may have a potential role for more precise diagnosis. Currently, bioinformatics methods play an important role in designing the antigenic multi-epitope to develop a serological diagnosis kit. In the current study, liner and conformational B-cell epitopes prediction of Dense granule protein 7 (GRA7), Surface Antigen 1 (SAG1), and Rhoptry protein 1 (ROP1) antigens have conducted by the ABCpred, IEDB, LBtope, CBTOPE and DiscoTope servers, respectively. Prediction of the 3D structure of three toxoplasma proteins(SAG1,GRA7,ROP1) and multi-epitope (final structure) was performed using -TASSER server. Model 1 was selected as the best model with C-Score: -3.89. The modeled GRA7, SAG1, and ROP1 structure and multi-epitope structure were validated by the Ramachandran plots Prosa and Verify 3D server. Epitopic regions of 3D protein structure were identified by Chimera UCSF software. Physicochemical properties of the Multi epitope were evaluated using ProtParam and SOLpro server. The synthesized multi-epitope was directly cloned into a pET28b expression plasmid and transformed into Escherichia coli BL21 (DE3) and induced by 1 mM IPTG. The expressed protein was finally confirmed by SDS-PAGE and dot-blot techniques. SDS-PAGE results of recombinant multi-epitope peptide (rMEP)-pET-28b showed a band of about 24.088 kDa. The western blot results indicated that rMEP had a high response with 1:50 with positive serum. The current results indicated that the integrated recombinant (SAG1, GRA7, and ROP1) multi-epitope antigens of T. gondii might be useful to develop practical diagnostic kits for acute and chronic toxoplasmosis.