Immunogenicity of dominant epitopes of complex antigen rhoptry protein 2-sunface protein 1 derived from Toxoplasma gondii

Abstract

Objective To analyze the immunogenicity of dominant epitope of complex antigen of rhoptry protein 2 (ROP2) and major surface protein 1 (SAG1) derived from Toxoplasma gondii (T. gondii). Methods Dominant epitope of ROP2-SAG1 containing both dominant T- and B-cell epitopes was predicted and selected from T. gondii with bioinformatics methods. The gene fragment cloned into pET32a expression vector was transformed into the competent cell (Escherichia coli strain Rosetta) and expressed under the induction. The protein purified by nitrilotriacetic acid (Ni-NTA) agarose resin were finally identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Japanese rabbits were immunized subcutaneously with purified epitope protein in contrast with control group immunized with pET32a protein or phosphate buffevred saline (PBS). The sera from immunized rabbits were collected at week 0, week 2 and week 4 for determination of epitope-specific antibody IgG using enzyme-linked immunosorbent assay, and immunodot assay was used to further confirm the specificity of antibody. After BALB/c mice were immunized with purified epitope proteins, the capacity of production of interferon-γ(IFN-γ) in splenocytes was detected by enzyme-linked immunospot assay. Results Relative molecular weight 30 000 of dominant epitope was derived from prokaryotic system. Then the rabbits immunized with purified dominant epitope could produce corresponding epitope-specific antibody IgG. And with the increased frequency of immunization, the level of antibody gradually increased. At week 2 and 4, higher antibody response were observed in group of rabbit immunized with dominant epitope than those of control group (1.454±0.098 vs 0.616±0.084, F=0.000, P<0.05; 2.299±0.224 vs 1.580±0.192, F=0.112, P<0.05). The antibody titer at week 4 was as high as 1∶40 960. Immunodot assay further confirmed the antibody specificity against the dominant epitope. The level of IFN-γ in splenocytes from mice immunized with dominant epitope after stimulation with three epitope specific CTL peptides (epitope peptides 1 [19.333±1.528]/100 000 cells, epitope peptides 2 ([40.333±1.528]/100 000 cells), epitope peptides 3 ([70.667±1.890]/100 000 cells) was significantly higher than that of PBS control group (epitope peptides 1 [1.033±0.150]/100 000 cells, epitope peptides 2 [1.045±0.110]/100 000 cells, epitope peptides 3 [1.041±0.120]/100 000 cells, F=0.284, 0.000 and 0.284, respectively; all P<0.05). The level of IFN-γ from splenocytes stimulated with combined peptide with cytotoxic T lymphocyte (CTL) epitope peptide and helper T cell epitope peptide (epitope peptides 3) was significantly higher than that with single CTL epitope peptides (epitope peptides 1 and epitope peptides 2, F=5.796 and 0.000, respectively; both P<0.05). Conclusion Screened dominant epitopes of ROP2-SAG1 from T. gondii derived from prokaryotic expression system exhibit remarkable immunogenicity. Key words: Toxoplasma; ROP2-SAG1; Epitopes; Immunogenicity