The intermediate washing steps with PBS-T and developing were as described earlier using AP substrate. Background was assessed by incubating the wells with the non-induced periplasmic extract. All assays were conducted in duplicate. We used a rapid dot-immunoblotting protocol. Volumes of 10 μl of sera samples from Toxoplasma sero-negative and sero-positive

patients selleck kinase inhibitor were spotted onto nitrocellulose membrane, allowed to air dry and then blocked with blotto. The membrane was incubated for 1 h at room temperature with a periplasmic preparation containing the SAG1–AP fusion protein. Specific immunocomplexes were detected by incubation for 20 min in the BCIP/NBT AP substrate buffer. The membrane was washed three times with PBS-T between each step. Background Gemcitabine price was assessed in the same conditions with the non-induced periplasmic extract. According to the primers used, sag1

coding gene fragment was PCR-amplified as 867 bp including SfiI/NotI clamp sequences (data not shown). After digestion with restriction enzymes, DNA fragment was ligated into the SfiI/NotI cloning site of the pLIP6-GN vector. The recombinant plasmid was transformed into the E. coli DH5α strain; rapid visual screening on BCIP containing agar plates allowed detection of recombinant clones and the corresponding plasmids were sequenced. As expected in all blue colonies, insertion of the sag1 gene between codons + 6 and Galactosylceramidase + 7 of AP gene restored the initial frame of the AP gene in the vector. In the retained plasmid, nucleotide and deduced amino acid sequences of sag1 were in agreement with GenBank database (accession no. X14080) (data not shown). The recombinant pLIP6-sag1–AP vector was subsequently used to transform E. coli XL1-blue strain. The colonies were grown in LB medium at 37 °C, and then induced with 0.5 mM IPTG at 28 °C overnight. Periplasmic fusion protein was extracted using cold osmotic shock. A protein band with an apparent molecular weight of 78 kDa

was detected after SDS-PAGE on homogenous 10% silver staining gel ( Fig. 1A, lane 2), in agreement with the SAG1–AP predicted molecular mass. This band was absent in the non-induced cell culture. The identity and the integrity of this band as the SAG1–AP conjugate were confirmed further by two Western blotting after SDS-PAGE. The first was revealed with the anti-bacterial AP MAb ( Fig. 1B) and detected the 78 kDa-recombinant protein in periplasmic and cytoplasmic fractions from induced recombinant bacteria tested. The second blot was revealed with the conformational anti-T. gondii SAG1 Mab ( Fig. 1C) and only the periplasmic SAG1–AP was detected. This means that the intact SAG1–AP fusion protein was released in soluble form into the bacterial periplasm, where the SAG1 antigen adopts a native-like structure. No visible degradation products are revealed using anti-SAG1, suggesting the stability of the fusion protein.