The resulting collection of homologous sequences was used to investigate the conservancy level of each predicted epitope at IEDB (Immune Epitope Database)28 using the epitope conservancy analysis tool (http://tools.iedb.org/conservancy). Multi-epitope vaccine candidate construction To generate the multi-epitope vaccine construct, the selected candidate epitopes, including 5 linear B-cell epitopes linked to each other using GPGPG and a total 5 high-affinity HTLs epitopes linked together AYY linkers, respectively. Antigenicity and allergenicity of Mouse monoclonal to C-Kit the vaccine construct The ANTIGENpro and VaxiJen 2.0 servers calculated the whole vaccine sequence antigenicity with the adjuvant sequence values of?0.92630 and 0.6976?with a threshold of 0.5 for the?parasite model, respectively. The allergenicity of the vaccine was predicted using the AlgPred and AllerTOP v. two servers and resulted in nonallergenic. Secondary structure prediction The predicted secondary structure using PSIPRED data was shown 60% alpha-helix, 39% beta-strand, and 0.4% coil in the final protein vaccine (Fig.?2). This secondary structure was used for refining the protein tertiary structure. Open in a separate window Figure 2 The PSIPRED server predicted a graphical demonstration of secondary structure properties of the final designed vaccine. Our vaccine’s protein sequence comprised 60% alpha-helices, 39% beta strands, and 0.4% coils. 3D structure homology modeling and validation SWISS-MODEL, Phyre2, and I-TASSER are the servers used for 3D structure modeling. In this study, c2ch7A_11 model was selected from the Phyre2 server (Fig.?3) as the best model according to preliminary validation analysis. In the chosen model, analysis with PROCHECK’s Ramachandran plot showed that 96% and 4% of residues are placed in favored regions and allowed, respectively (Fig.?4A). The ProSA z-score and ERRAT were???2.84 and 98.06, respectively (Fig.?4B) and (Fig.?4C). Open in a separate window Figure 3 The 3D model of the final designed vaccine was obtained after homology modeling on Phyre2. Open in a separate window Figure 4 The validation of the final 3D model. (A) PROCHECK’s Ramachandran plot illustrates that the residues are placed in the allowed (96%) and favored (4%) regions. (B) ProSA Z-score plot shows a -2.84 score in the range of conformation of the native protein. (C) ERRAT plot showed the overall quality factor to be 98.06%. Conformational (discontinuous) B-cell epitopes prediction In the 3D model of the final designed vaccine construct, residues with a value of 0.7 or higher were identified as conformational epitopes (Table ?(Table4).4). Also, discontinuous epitopes predicted in the 3D structure of the final multi-epitope construct are shown (Fig.?5). Table 4 Predicted conformational epitopes of the final designed vaccine by the ElliPro server. K12Finally, using and restriction sites, 1242 nucleotides as the Cintirorgon (LYC-55716) optimized sequence was cloned into the pET28a vector. The 6xHis-tag at the C-terminal of the multi-epitope protein vaccine was placed for the purification process (Fig.?9). Open in a separate window Figure 9 In silico cloning of multi-epitopes vaccine sequence into pET28a (+) expression vector using SnapGene software free-trial (https://www.snapgene.com/free-trial/), the red and gray semicircles represent the multi-epitopes vaccine sequence and the pET28a (+) backbone, respectively. In silico immune responses simulation against the designed Vaccine The immune Cintirorgon (LYC-55716) responses profile of the designed vaccine is shown in Fig.?10A. The combined IgM?+?IgG titer remained at about 680,000?xx/mL; the IgM titer alone was calculated to be around 530,000?xx/mL, and the combined IgG1?+?IgG2 titer was about 150,000?xx/mL. These data show that the titer of immunoglobulins increased after the injection of the designed vaccine (as antigen) with a marked decrease in the antigen concentration. Analysis of interleukins (IL) and cytokines production showed high titers of Cintirorgon (LYC-55716) IFN-g and IL-2, indicating that the antigen (designed vaccine) could trigger a strong and stable response. (Fig.?10B). B-cell populations with a significant increase in the memory, non-memory cells, and IgM isotype were predicted (Fig.?10C). The T-helper cell population per state (cells per mm3) represents increased levels after the injection (Fig.?10D). Open in a separate window Figure 10 In silico immune simulation results of the designed vaccine from C-ImmSim server. (A) The titer of immunoglobulins was produced after the injection of the designed vaccine. (B) High titers of IFN-g and IL-2 were induced after vaccine administration. (C) B-cell populations prediction with Cintirorgon (LYC-55716) a significant increase in the memory, non-memory cells, and IgM isotype. (D) The T-helper cell population per state (cells per mm3) levels increased after the injection. Materials and methods Retrieving of PfGARP and flagellin protein sequences In this research, glutamic acid-rich protein (PfGARP) (P13816) (UniProt database at http://www.uniprot.org/) was selected as a parasite antigen. It.