Toxoplasma gondii is an important pathogen in veterinary and human medicine. In this study, a new multiplex TaqMan real-time PCR for detection of T. gondii DNA was developed. This assay consisted of new sets of primers and probes which targeted B1 gene and ITS-1 region of T. gondii, with Vibrio cholera gene as internal control. The B1 gene primers were designed to detect T. gondii RH strain, while the ITS-1 region primers detected most T. gondii strains. Specificity test using common protozoal and bacterial DNA revealed that the assay was very specific to T. gondii. Standard curves constructed using human body fluids spiked with T. gondii (RH and ME49 strains) showed that the sensitivity of the assay was one parasite, with R² value of 0.975 to 0.999 and efficiency of 97% to 99% for all types of samples. The assay performed on DNA extracted from tissues of mice infected with T. gondii showed that liver contained the highest parasite load for both strains of T. gondii. The multiplex real-time PCR developed in this study would be potentially useful for detection of T. gondii in human and animal samples.
GRA4 of Toxoplasma gondii has been shown to prompt IgG, IgM and IgA responses in previous studies and is thus considered one of the major immunogenic proteins from T. gondii that can be used for both diagnostics purposes and vaccine development. This study seeks to clone and express the GRA4 in Pichia pastoris, which has numerous advantages over other systems for expression of eukaryotic proteins. In order to achieve this, the gene was cloned into the pPICZα A expression vector, which was then incorporated into the P. pastoris genome via insertional integration for expression of the recombinant protein, under the AOX1 promoter. The antigen was expressed along with the prepro sequence of the α-factor of yeast so that it could be excreted out of the P. pastoris cells and obtained from the medium. Upon SDS-PAGE analysis it was found that the recombinant protein was expressed optimally as a 40 kDa protein after 96 hours of induction with 0.75% of methanol. The expressed GRA4 protein showed discrepancy in size with the calculated molecular mass. This may be attributed to the various posttranslational modifications including glycosylation and phosphorylation. Despite the difference in molecular weight, the recombinant protein was able to detect toxoplasmosis in Western blot format. The recombinant GRA4 was expressed with an intact polyhistidine-tag, which could be used for future purification of the antigen.
Toxoplasma gondii is an obligate intracellular zoonotic parasite of the phylum Apicomplexa which infects a wide range of warm-blooded animals, including humans. In this study in-vivo induced antigens of this parasite was investigated using in-vivo induced antigen technology (IVIAT) and pooled sera from patients with serological evidence of acute infection.
In this study, we successfully expressed a chimerical surface antigen 1 and 2 (SAG1/2) of Toxoplasma gondii in Pichia pastoris. Eighty human serum samples, including 60 from confirmed cases of toxoplasmosis, were tested against the purified recombinant SAG1/2 in Western blots. Results of Western blots targeted at Toxoplasma IgG and IgM showed that the recombinant SAG1/2 reacted with all sera from the toxoplasmosis cases but none with the Toxoplasma-negative serum samples. These results showed that the P. pastoris-derived recombinant SAG1/2 was sensitive and specific and suitable for use as antigen for detecting anti-Toxoplasma antibodies. To further investigate the immunological characteristic of the recombinant protein, the recombinant SAG1/2 was injected subcutaneously into BALB/c mice, and their serum was tested against total protein lysate of T. gondii. Mice immunized with the recombinant SAG1/2 reacted specifically with the native SAG1 and SAG2 of T. gondii. Significant proliferation of splenocytes stimulated with tachyzoite total protein lysate was observed in vaccinated BALB/c mice but not in those from negative control mice. Specific production of IFN-γ, the Th1-type cytokines, was also found in stimulated splenocytes from vaccinated mice. These results show that the chimeric protein recombinant SAG1/2 can elicit a Th1-associated protection against T. gondii infections in mice. Finally, vaccinated mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P
The full length surface antigen 2 (SAG2) gene of the protozoan parasite Toxoplasma gondii was cloned and intracellularly expressed in the Pichia pastoris expression system. The molecular weight of the expressed recombinant SAG2 (36 kDa) was much larger than the native SAG2 (22 kDa). This discrepancy in size was due to hyperglycosylation, as deglycosylation assay reduced the size of the recombinant SAG2 to 22 kDa. Despite being hyperglycosylated, the recombinant SAG2 reacted strongly with pooled anti-Toxoplasma human serum, pooled anti-Toxoplasma mouse serum and a SAG2-specific monoclonal antibody. The glycosylated recombinant SAG2 was further evaluated in Western blot and in-house enzyme-linked immunosorbent assay (ELISA) using 80 human serum samples, including confirmed early acute (IgM positive, IgG negative; n=20), acute (IgM positive, IgG positive; n=20) and chronic (IgM negative, IgG positive; n=20) toxoplasmosis patients, and toxoplasmosis negative control patients (n=20). Results of the Western blot showed that the recombinant SAG2 reacted with all 60 samples of the toxoplasmosis cases but not with the Toxoplasma-negative samples. The sensitivity of in-house ELISA was 80%, 95% and 100% for early acute, acute and chronic patients' serum samples, respectively. Vaccination study showed that serum from mice immunised with the glycosylated recombinant SAG2 reacted specifically with the native SAG2 of T. gondii. The mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P<0.01) and their survival time was increased compared to controls. Therefore, the present study shows that the P. pastoris-derived recombinant SAG2 was specific and suitable for use as antigen for detecting anti-Toxoplasma IgG and IgM antibodies. The vaccination study showed that recombinant SAG2 protein was immunoprotective in mice against lethal challenge.