Female6 to 8 week old BALB/C mice were purchased from Pasteur Institute of Iran. Allexperimental procedures were conducted according to animal guidelines ethics.The study was approved by the Ethics Committe of Iran university of medicalsciences. (Approval No..9221577201 )2.2.
Toxoplasma gondii strainsThehigh virulence T.gondii RH strain was provided by the toxoplasmosisresearch center from Tehran university of medical sciences, Iran. BALB/c micewere injected via peritoneal cavity with 1×105 of tachyzoites suspended in sterilePhosphate-Buffered Saline (PBS; pH=7.4) comprising 100 mg/ml streptomycin and 100IU/ml penicillin. Tachyzoites were harvested after 3-4 day from peritoneal cavity of mice. (GRA14).2.
3.Preparation of Toxoplasma Lysate Antigen (TLA)Thetoxoplasma lysate antigen (TLA) was set up as described previously (ROP18 Rop5). Briefly, Peritonealcells were washed with PBS and centrifuging at 800×g for10 min at 4°C. thepurified tachyzoites were disrupted by ten cycles of freezing and thawing inliquid nitrogen. Lysate was centrifuged at 10000g for 20 min at 4°C thenfiltered with 0.22 ?m sterile filter. The total protein was assessed by BCA (bicinchoninicacid) method.
TLA specimen were aliquoeted and stored at -80°C.2.4.
Bioinformatic implements forpredicting T-cell epitopes T cell epitopes of ROP2, GRA4 and AMA1of T. gondii RH strain were predicted with bioinformatics softwares ofIEDB. Among the predicted epitopes, the 8 T-cell epitopes and 2 MHC II fragments were chosen due a higher score ofmentioned genes related to a higher possibility of the residue being involvedin the epitope, using Database online serviceIEDB(http://tools.immuneepitope.org/mhcii) (Table 1).2.5. predicting B-cell binding epitopeBcell epitope of SAG1were chosen in previous study by A.
Stuara (). The epitope detectd at the surface ofthe D1 domain from monomeric form of SAG1 molecule that compound to a Fabmonoclonal antibody. This antibody increase against tachyzoite strongly. So, this epitope is an immunogoldregion on the surface of SAG1.Multi-epitopes gene synthesis Bcell epitope of SAG1 was linked to T cell epitopes with GGGGS linker encoding amino acids. Consequently theToxoplasma gondii multi-epitopes gene (TgMEG) was engendered usingchemical gene synthesis. TgMEG also comprised an enzyme restriction Nde1 sequence, AAT start codon, following aTAA stop codon and an enzyme restriction Xho1 sequence and with double six histidine residues tag(6×His).
TgMEG, a total of 700 bp, wassynthesized and cloned in the pET28avector by Pepmic Co, Suzhou China.2.5.Expression and Purification of recombinant proteinTheE. coli strain of BL21 (DE3) (Novagen, Darmstadt, Germany) cells weretransformed with pET28b/MEG and were grown in LB broth medium with 100mg/mlkanamycin to an optical density of 0.6 at 600nm. Enhance the solubility oftarget protein with 0.
5 mM of IPTG (Isopropyl?-D-1-thiogalactopyranoside) (Sinaclon, Iran) at 37°C, followed by harvest ofthe bacterial cell by centrifugation at 4000 rpm for 30 at 4°C and lysis. Theexpression products were disrupted by freeze-thawing and centrifugation at 13,000g, 4°C for 20 min. proteins were produced in insoluble forms accumulated ininclusion bodies, as estimated by 12% SDS-PAGE electrophoresis, they wereisolated under denaturing conditions with 6M urea. The solubilized proteins were purified by affinityNi2+-chromatography (Qiagen, Hamburg, Germany), according to the manufacturer’sinstructions.
After purification the recombinant proteins were refolded, analyzedby SDS-PAGE electrophoresis. Protein Ladder (Sinaclon, Iran) were used as a molecularweight marker for SDS-PAGE. Finally, the purified protein concentration wasassessed by a BCA protein assay reagent.