Evaluation of Pichia pastoris–Expressed Recombinant Rhoptry Protein 2 of Toxoplasma gondii for Its Application in Diagnosis of Toxoplasmosis

Abstract

Rhoptry protein 2 (ROP2) of Toxoplasma gondii is a rhoptry-secreted protein that plays a critical role in parasitophorous vacuole membrane formation during invasion. In previous studies, ROP2 has been shown to be efficient in triggering humoral and cell-mediated responses. High immunogenicity of ROP2 makes it a potential candidate for diagnosis and vaccination against toxoplasmosis. In this study, the ROP2 gene was cloned into pPICZα A expression vector and extracellularly expressed in the yeast Pichia pastoris, which has numerous advantages over other expression systems for eukaryotic proteins expression. The effectiveness of the secreted recombinant ROP2 as a diagnosis agent was assessed by Western Blot with 200 human serum samples. Recombinant ROP2 reacted with toxoplasmosis-positive human serum samples and yielded an overall sensitivity of 90% and specificity of 95%. However, recombinant ROP2 is a better marker for detection of IgG (91.7%) rather than IgM (80%).

Toxoplasma gondii Recombinant Antigens as Tools for Serodiagnosis of Human Toxoplasmosis: Current Status of Studies

“…In addition, these proteins do not contain contaminants derived from the bacterial cells and, therefore, do not give cross-reactions with human sera. In recent years, the Pichia expression system in particular has been used successfully to produced numerous T. gondii recombinant antigens (22,24,75,(80)(81)(82)(83). Thus, there is a need to obtain the new recombinant antigens in different eukaryotic expression systems.…”

Evaluation of a recombinant rhoptry protein 2 enzyme-linked immunoassay for the diagnosis of toxoplasmosis acquired during pregnancy

“…ROP2 has been cloned and expressed in vitro and may thus be used for the diagnosis of and vaccination against toxoplasmosis. Indeed, IgA, IgM and IgG anti- T. gondii antibodies in human toxoplasmosis can be detected by using rROP2 expressed in E. coli ( Chang et al 2011 , Yan et al 2012 ). An adequate selection of recombinant antigens may also be used in serological tests to differentiate between recently and previously acquired infections.…”

“…Using WB on samples from patients with toxoplasmosis, amebiasis, cysticercosis, filariasis, malaria or toxocariasis, the sensitivity and specificity for diagnosing toxoplasmosis with rROP2 was 90% and 95%, respectively ( Chang et al 2011 ). In that study, rROP2 was better at detecting IgG (91.7%) than IgM (80%) ( Chang et al 2011 ). We did not perform WB, which is likely to have a different sensitivity and specificity compared with the ELISA-rROP2.…”

Toxoplasmosis: IgG Avidity and Its Implication in Serodiagnosis

“…rROP1 has shown its diagnostic value in IgG ELISA avidity tets for identification of acute infections (Holec-Gąsior et al, 2009, 2010a. There is data regarding the potential use of rROP2 for the diagnosis of acute toxoplasmosis that have focused on IgG reactivities (Martin et al, 1998;Chang et al, 2011). In a mouse model, IgM antibody against rROP4 was significantly higher than IgG antibodies with a peak of detection coming on the turn of the acute to a latent infection (Gatkowska et al, 2010).…”

“…They are exocytosed upon host cell invasion and their contents are involved in many functions fundamental for the parasite to enter into host cells, and for the establishment and maintenance of the parasitophorous vacuole membrane and acquisition of nutrients (Dubremetz, 2007). The use of recombinant ROP antigens (rROP) has been largely described as the antigenic substrate to use in ELISA tests to detect infection with T. gondii (vanGelder et al, 1993;Aubert et al, 2000;Chang et al, 2011). rROP1 has shown its diagnostic value in IgG ELISA avidity tets for identification of acute infections (Holec-Gąsior et al, 2009, 2010a.…”