Expression and single-step purification of GRA8 antigen of Toxoplasma gondii in Escherichia coli

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Publisher: Avicenna Research Institute
Document Type: Medical condition overview
Length: 4,763 words
Lexile Measure: 1410L

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Diagnosis of Toxoplasma gondii(T.gondii) infection is of great medical importance especially for pregnant women and immunosuppressed patients. Numerous studies have shown that the recombinant production of several toxoplasma antigens, including dense granule antigens (GRAS) has a great potential as diagnostic reagents. Previous studies reported expression of amino terminal GRA8 protein in fusion with large tags. In the present study, we produced truncated GRA8 (GRAS), excluded from the signal peptide and C-terminal transmembrane domain, with a short fusion tag in Escherichia coil GRA8 was purified using an optimized single-step Immobilized Metal ion Affinity Chromatography (IMAC). The purity and yield of GRA8 was highest at pH=9.25. At this pH, 13.6 mg of GRA8 was obtained with the purity of 97.97%. Immunogenicity of the protein was evaluated in Western blot analysis showing the serum sample from a rabbit immunized with GRAS recognized a single antigen of T.gondii tachyzoite at the expected molecular weight of native GRA8. To diagnosis acute toxoplasma infection in pregnant women, an indirect immunoglobulin M (1gM) enzyme-linked immunosorbent assay (ELISA) was developed using GRA8 resulting in a test specificity and sensitivity of 97.1% and 60.6%, respectively. These results demonstrated that immunogenic GRA8 can be produced in fusion with a short tag and purified near to homogeneity using an optimized IMAC. GRA8-IgM-ELISA was useful for detection of acute toxoplasma infection.

Avicenna J Med Biotech 2011;3(2):67-77

Keywords: Enzyme-linked immunosorbent assay, Gene expression, GRA8 protein, immunoglobulin M, Toxoplasma gondii


Toxoplasmosis is caused by the protozoan parasite Toxoplasma gondii in a large variety of domestic and wild mammals and birds including human being (1). The infection generally is asymptomatic in immunocompetent individuals. However, in immunocompromised patients, such as those with HIV/AIDS, infection can cause life-threatening encephalitis and can be fatal if not recognized and treated soon (2). Furthermore, since the parasite can cross the placenta, the primary maternal infection with T.gondii can be transmitted to the fetus and may lead to severe congenital defects such as hydrocephaly, mental retardation, chorioretinitis, visual impairment or even death in utero (2-5). In livestock, abortion of ewes causes considerable economic losses (6).

Accurate diagnosis of primary toxoplasma infection and subsequent chemotherapy reduce the risk of congenital infection (2), (3). Detection of toxoplasma infection and distinction between acute and chronic phases of the infection are mainly based on serological tests enabling the detection of anti-toxoplasma immunoglobulin G (IgG) and IgM antibodies in blood (3).

The conventional serological tests are primarily based on whole T.gondii antigens which might have inconstant quality and external antigens from mouse peritoneal cavity or cell culture. The use of these antigen preparations makes standardization of the assays difficult and also results in insufficient specificities for differentiation between acute and chronic infection (3), (7), (8). The recombinant toxoplasma antigens, which can be easily produced in large amounts and with constant quality, are the good candidates for replacement of the crude antigens. Development of serological tests using recombinant toxoplasma antigens which are representative of acute or chronic infection is a present trend in discrimination between...

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Gale Document Number: GALE|A304726992