Recombinant Dense Granular Protein (GRA5) for Detection of Human Toxoplasmosis by Western Blot

Abstract

Toxoplasma gondii infects all warm-blooded animals, including humans, causing serious public health problems and great economic loss for the food industry. Commonly used serological tests require costly and hazardous preparation of whole Toxoplasma lysate antigens from tachyzoites. Here, we have evaluated an alternative method for antigen production, which involved a prokaryotic expression system. Specifically, we expressed T. gondii dense granular protein-5 (GRA5) in Escherichia coli and isolated it by affinity purification. The serodiagnostic potential of the purified recombinant GRA5 (rGRA5) was tested through Western blot analysis against 212 human patient serum samples. We found that rGRA5 protein was 100% specific for analysis of toxoplasmosis-negative human sera. Also, rGRA5 was able to detect acute and chronic T. gondii infections (sensitivities of 46.8% and 61.2%, resp.).

Development and Evaluation of Recombinant GRA8 Protein for the Serodiagnosis of Toxoplasma Gondii Infection in Goats

“…After removing the transmembrane region, the gene fragment encoding amino acids 24-194 was used to express the protein, and a speci c 27-kDa band was observed on SDS-PAGE. Our result was similar to that of Babaie et al [11], who designed and expressed a recombinant protein from the GRA8 gene fragment corresponding to amino acids 23-169. However, a difference in size between the apparent band and the calculated molecular weight of TgGRA8 (22.62 kDa) was observed in the present study.…”

“…Dense granule antigens (GRAs) of T. gondii are secreted in the parasitophorous vacuole (PV), and they are involved in survival and virulence of the parasite [9]. Several studies demonstrated the diagnostic potential of numerous GRAs such as GRA2 [10], GRA5 [11], GRA6 [12], and GRA7 [13,14]. GRA8 is a 38-kDa praline-rich (24%) protein that is released from PVs shortly after invasion.…”

Development and evaluation of recombinant GRA8 protein for the serodiagnosis of Toxoplasma gondii infection in goats

“…After removing the transmembrane region, the gene fragment encoding amino acids 24-217 was used to express the protein, and a speci c 27-kDa band was observed on SDS-PAGE. Our result was similar to that of Babaie et al [12], who designed and expressed a recombinant protein from the GRA8 gene fragment corresponding to amino acids 23-169. However, a difference in size between the apparent band and the calculated molecular weight of TgGRA8 (23.71 kDa) was observed in the present study.…”

“…Dense granule antigens (GRAs) of T. gondii are secreted in the parasitophorous vacuole (PV), and they are involved in survival and virulence of the parasite [10]. Several studies demonstrated the diagnostic potential of numerous GRAs such as GRA2 [11], GRA5 [12], GRA6 [13], and GRA7 [14,15]. GRA8 is a 38-kDa praline-rich (24%) protein that is released from PVs shortly after invasion.…”

Effective Diagnostic Marker for Serodiagnosis of Toxoplasma gondii Infection: New Developments and Perspectives

“…The potential ability of specific T. gondii recombinant antigen to identify the clinical phases of the disease was extensively studied, in the attempts to discriminate acute from chronic infection. The performance of recombinant antigen seems to be sensitive enough and promising in differentiating acute versus chronic infection [35,38]. Although there is no clear definition of either chronic or acute infection, identification of recently acquired infection normally relies on the detection of specific IgM in patients serum;…”