The RH strain of T. gondii was maintained by peritoneal passage in Balb/c mice. Prior to use, tachyzoites were purified by centrifugation over 40% Percoll (Amersham Phamacia Biotech, Uppsala, Sweden) in PBS solution (Sohn and Nam, 1999).

Proteins were resolved by 12% SDS-PAGE under reducing conditions and western blotted as described in Ahn et al. (2001). Nitrocellulose sheets blocked by 5% skim milk in PBS/0.05% Tween 20 (PBS/T) were incubated with 1:1000 diluted mAb, and then with 1:2000 diluted HRP-conjugated goat anti-mouse IgG antibody (Cappel, Costa Mesa, CA, USA). They were soaked in enhanced chemiluminescence (ECL) solution (Amersham Phamacia Biotech) for 1 min and exposed to an X-ray film (Konica, Tokyo, Japan).

Free tachyzoites were attached to 18 mm cover slips by using a cytospin. Tachyzoites were fixed with cold absolute methanol for 5 min. Vero cells (CRL 6318, American Type Culture Collection, Rockville, MD, USA) cells were maintained in DMEM supplemented with 10% FBS (Gibco BRL, Rockville, MD). Cells cultured on 18 mm coverslips in 24-well plates were infected with tachyzoites. Cells were fixed either with cold absolute methanol for 5 min or with 3% formaldehyde for 10 min and then permeabilized by 0.05% Triton X-100 for 5 min, separately. MAb was diluted in 1:100 of 3% BSA/PBS and FITC-conjugated goat anti-mouse IgG antibody (Sigma Chem Co., St. Louis, MO, USA) was used in 1:500. Fluorescence was observed under a fluorescence microscopy (Axiophot, Carl Zeiss Co., Oberkochen, Germany).

A T. gondii λZAPII cDNA expression library was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (McKesson Biosciences, Rockville, MD) and screened in E. coli XL1-Blue MRF' (Stratagene, La Jolla, CA) using mAb in PBS/T containing 1% (w/v) BSA. Bound antibody was detected using the ECL Detection System (Amersham Phamacia Biotech). Positive plaques were recovered and rescreened by the same procedure. pBluescript SK phagemids were isolated by co-infection of the λZAPII phage and ExAssist helper phage (Stratagene). Excised phagemids were further propagated in the E. coli SOLR host strain (Stratagene). Phagemid DNA was purified from single colonies using the Wizard Plus SV Miniprep kit (Promega, Madison, WI).

All DNA sequencing was performed using dye terminator fluorescent-based sequence analysis on an Applied Biosystems 373 automated sequencer. The ends of the cDNA clones were sequenced using primers against the vector T7 and T3 promoter sequences. All other sequencing primers were custom synthesized (Bionia Co., Daejon, Korea). Sequences of cDNA clones were used to search for homologous sequences in the Toxoplasma dbEST (Database of Expressed Sequence Tags) using the BLASTn algorithm with the default settings. The protein sequences were compared to the GenBank database using BLASTp. PROSITE was used to search for motifs and post-translational modification sites.

Total T. gondii tachyzoite RNA was extracted using Tri reagent (Sigma Chem Co.) according to the manufacturer's instructions. The 5' untranslated region was amplified using the 5'-RACE procedure (Frohman et al., 1988). First strand cDNA was synthesized from 1 µg of total RNA using the Superscript Preamplification System (Life Technologies, Gaithersburg, MD). DeoxyCTPs were added to the 3' end of the non-coding cDNA using terminal deoxynucleotide transferase (Life Technologies). PCR amplification of C-tailed cDNA was performed with an anchor primer (5'-CTA ATA CGA CTC ACT ATA GGG CAA GCA GTG GTA TCA ACG CAG AGT-3'; Bionia Co.) and a gene-specific primer (5'-AAG CAG TGG TAT CAA CGC AGA GT-3'). First round product was further amplified with the abridged universal anchor primer (5'-CTA ATA CGA CTC ACT ATA GGG C-3') and an internal gene-specific primer. Second round PCR product was cloned into the pGEM-T EASY vector (Promega) and sequenced.

RT-PCR was performed to amplify the DNA fragments of open reading frame of the protein with a forward primer of 5'-GGA TTC GCG GGT CCC AAG GGA A-3' and a reverse primer of 5'-AAG CTT GAC TCC GAC TTT TGC GCG-3' directed against the sequence. Amplified DNA was subcloned into pET-28a vector (Novagen, Madison, WI) in BamH I and Hind III sites to transform BL21 (DE3, Stratagene). Recombinant protein was obtained by IPTG induction and purified through the Ni-NTA column. The protein was used as antigen in western blot against 23 sera from toxoplasmosis patients (Choi et al., 1997; Kim et al., 2000) and high risk pregnancy women that reacted with recombinant SAG1 antigen (SAG1-positive, Nam et al., 1996) and normal control (SAG1-negative).

In the course of screening for mAbs against crude extract of T. gondii tachyzoites, we identified a hybridoma supernatant (Tg621) that reacted with 38 kDa antigen on western blot. The hybridoma was expanded, cloned and rescreened. The ascitic fliud of mouse included the corresponding mAb and blotted 38 kDa protein on western blot of whole parasite extract of T. gondii under reducing condition (Fig. 1). The mAb precipitated protein of the corresponding size from whole extract of Neospora caninum, a neighboring apicomplexan parasite. By immunofluorescence microscopy with mAb Tg621, fluorescence was dispersed in the cytoplasm of the extracellular tachyzoites (Fig. 2, A and B) and similar pattern in the intracellular growing tachyzoites without involvement to parasitophorous vacuolar membrane (Fig. 2, C and D).

The mAb Tg621 was used to screen a T. gondii λZAPII cDNA expression library. Screening of the expression library resulted in the isolation of cDNA clone, which were isolated from the pBluescript SK phagemids. The nucleotide sequence of the clone contained a poly(A⁺) tail. The complete 5' untranslated sequence, determined by 5'-RACE, yielded a cDNA which spanned a total of 1,592 bp and included a predicted open reading frame of 942 bp. A search of the Toxoplasma database of expressed sequence tags identified a contig assembly containing 30 expressed sequence tags with a high BLAST score. Full cDNA sequence was enrolled to GenBank (accession number AY217739) as a ribosomal P protein of T. gondii, hereafter referred to as TgRPP. Using the first in-frame ATG as the start site downstream of consensus sequence of protozoal/toxoplasmal translation initiation, TgRPP is predicted to encode a polypeptide of 313 amino acids with a molecular mass of 38 kDa (Fig. 3). Following the first ATG was no hydrophobic region that constituted a potential signal sequence. The predicted protein encoded by TgRPP had a domain with remarkably high hydrophobic alanines and acidic glutamic acids in C-terminal end. A PROSITE search yielded no obvious sequence motifs or post-translational modification sites within the TgRPP sequence.

The deduced amino acid sequence of TgRPP was compared to the GenBank database using BLASTp, which resulted in so many homologous sequences with a high BLAST score. Clustal W alignment of apicomplexan ribosomal P proteins was described in Fig. 4 with reference to those of mouse and human. Amino acid sequence of TgRPP showed 68% homology with that of Eimeria tenella, 58% with Babesia bovis, and 51% with mouse and 17% with human ribosomal P protein.

DNA fragment of ORF with several base pairs of 3'-UTR was amplified by RT-PCR as 1,013 bp as designed in Fig. 5. Amplified DNA was subcloned into pET-28a vector to transform BL21 (DE3). Recombinant protein was obtained by IPTG induction and purified through the Ni-NTA column as 43 kDa protein containing TgRPP and 6x His Tag, thrombin, and T7 Tag in N-terminal of insert (Fig. 6A). Recombinant TgRPP and naive TgRPP in tachyzoite extracts were blotted by mAb Tg621 as 43 and 38 kDa bands, respectively (Fig. 6B).

Recombinant TgRPP was used as antigen in western blot against the sera of toxoplasmosis patients and normal control with reference to recombinant SAG1 antigen. Sera of 17 among 23 SAG1-positive detected TgRPP (74.0%) whereas 3 sera in 36 SAG1-negative reacted with TgRPP (8.3%).