Tachyzoites of virulent RH strain of T.gondii were injected into peritoneal cavity of Swiss mice. Three days later, tachyzoites were harvested, washed with phosphate-buffered saline (PBS) and stored at -80 ^o C until used.

Escherichia coli DH5α (Invitrogen, Carls-bad, CA) strain bacteria were used for cloning purpose. The mammalian expression plasmid pcDNA3.1/Hygro (24) (Invitrogen, Carlsbad, CA) was used for expression of GRA5.

The GRA5 gene fragment encoding mature full-length protein, residue 27-120, was PCR- amplified from genomic DNA of T. gondii RH strain using the following oligonucleotide primers: Forward primer: 5' GCC ACC ATG GGT TCA ACG CGT GAC GTA GGG TCA G 3'; Reverse primer: 5' GCT GAG ACA ACA AAG AGC CGA GCA ACA CAG TGC 3'

The complete Kozak translational consensus sequence (underlined) was introduced in the beginning of the PCR product. PCR amplification was performed using the following conditions: 1 cycle of 95 °C for 5 min then 30 cycles of 95 °C for 30 sec, 58 °C for 1 min, and 72 °C for 30 sec. Final primer extension was extended to 30 min at 72 °C. PCR product was analysed by electrophoresis on 1.0 % agarose gel.

The PCR product was inserted into T/A cloning vector pTZ57R/T, excised by KpnI / ApaI enzymes, and subcloned into KpnI/ApaI restriction sites of pcDNA3.1/Hygro (24) plasmid. Selection of recombinant clone harboring GRA5 DNA was performed using PstI digestion of recombinant plasmids followed by sequence determination. The resulting plasmid was named pGRA5.

HEK 293-T cells, an embryonic kidney cell line, were grown at 37 °C in a humidified 5% CO2 atmosphere in 35-mm wells in Dulbecco's Modified Eagle Medium (DMEM) containing 100 U/ml each penicillin and streptomycin and 10% fetal calf serum, and were transfected at 50-70% confluency with 4 µg of pGRA5 or pcDNA3.1 using PolyFect transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer instructions. The transfected cells were incubated with PolyFect for 16 to 24 hr before replacing the medium with 2 ml of fresh medium. After 3 days, cell monolayers were washed 3 times with Phosphate Buffered Saline (PBS) and scraped into 1 ml of PBS. Cells were then recovered by centrifugation at 12,000 × g for 15 min and stored at -20 °C.

HEK 293-T cells were grown on 12-mm coverslips in 24-well plates and transfected with pGRA5 as described in the previous section. Three days after transfection, cells were fixed with 5% paraformaldehyde in PBS for 10 min, and permeabilized with 0.2% Triton X-100 in PBS for 3 min. Subsequently, cells were incubated with mAb anti-GRA5 TG17-113 (30), diluted 1:500 in 1% FBS in PBS for 1 hr at room temperature. Cells were washed with PBS and incubated for 1 hr with FITC-conjugated anti-mouse IgG (Sigma, St. Louis, MO, USA), diluted 1:20,000 in 1% FBS in PBS. Coverslips were mounted using Prolong antifade reagent (Molecular Probes) and observed using a Zeiss Axioplan II equipped for phase-contrast and epifluorescence microscopy. Photographs were taken at the magnification × 100 using a Zeiss Axiocam MRm coupled to the AXIOVISION 4.5 software and processed with ADOBE PHOTO-SHOP 6.0.

Western blot analysis of HEK 293-T cells transfected with pGRA5 was performed on cell pellets of single 35-mm wells. The pellet was resuspended in 50 µL of SDS-PAGE sample buffer, sonicated, boiled for 5 min and 15 l was loaded onto a 15% polyacrylamide gel. Proteins were transferred onto nitrocellulose membrane via electrophoresis, carried at 100 V and 350 mA for 1 hr, using Bio-Rad transfer system (Bio-Rad, Hercules, CA). The membrane was saturated for 1 hr with 5% fat-free dried milk in PBS and probed with the mAb anti-GRA5 diluted 1:1000 in saturation buffer. The membrane was incubated for 1 hr with peroxidase-conjugated goat anti-mouse IgG (Sigma, St. Louis, MO, USA) diluted 1:20,000 in saturation buffer, and signals were detected using super signal ECL (Enhanced Chemiluminescence) system (Pierce chemical, Rockford, IL).

The DNA encoding mature GRA5 antigen, residue 27-120 was amplified from genome of T.gondii RH strain by means of PCR. The complete Kozak consensus sequence, GCC ACC ATG G, was introduced in 5' of the GRA5 DNA. The specific product of 333 bp was observed in gel agarose electrophoresis (Figure 1). The PCR product was inserted into T/A cloning vector, pTZ57R/T, excised by KpnI/ApaI enzymes and subcloned into KpnI/ ApaI restriction sites of pcDNA3.1 mammalian expression plasmid. Screening of recombinant clones harboring pcDNA3.1-GRA5 (pGRA5) plasmid was performed by restriction digestion using PstI enzyme. Digestion of the recombinant plasmid produced two fragments of 4243 and 1626 bp, but nonre-combinant plasmid gave rise to only one DNA band (Figure 2). Sequence analysis of the cloned gene revealed 100% homology with the published sequence of gra5.

Expression of the recombinant protein by mammalian cells transfected with a plasmid DNA is required for stimulation of the immune system. As such expression of GRA5 protein was assessed in HEK 293-T cells transfected with pGRA5. Transfected cells were cultured for 3 days, washed, and expression of recombinant GRA5 was assessed by Western blotting and immunofluorescence staining. The result showed that HEK 293-T cells transfected with pGRA5 produced antigenic GRA5 protein (Figure 3). In Western blotting, recombinant GRA5 was detected as a band of about 18 kDa, which was about the same size as the native protein (Figure 3). In contrast, no immunoreactive material was found in the lysate of HEK 293-T cells transfected with pcDNA3.1 plasmid. The smaller and larger protein bands in the lane 4 are probably T.gondii antigens reacted non-specifically with the mAb anti-GRA5. Immuno-fluorescence staining showed HEK 293-T cells transfected with pGRA5 specifically produced GRA5 protein (Figure 4) which was located in the cytoplasm.