ABSTRACT
Toxoplasmosis is an infection caused by the protozoan parasite Toxoplasma gondii [T.gondii] throughout the world. Although usually asymptomatic, the infection can cause serious medical problems in immunocompromised individuals and fetuses. Toxoplasmosis also causes considerable economic loss because of abortion in livestock. DNA vaccination is a promising approach against intracellular parasites such as T.gondii. The goal of this study was to construct and evaluate functionality of a mammalian plasmid expressing GRA5 antigen of T.gondii as a possible DNA vaccine. GRA5 gene fragment devoid of the signal sequence, was amplified from genomic DNA of T.gondii RH strain, and cloned into pcDNA3.1 plasmid. The pcDNA3.1-GRA5 [pGRA5] was analyzed by restriction enzyme digestion followed by sequence determination. The pGRA5 was transfected into HEK 239-T human kidney cells, and expression of GRA5 antigen was investigated by Western blotting and immunofluorescence staining. The sequence encoding GRA5 was cloned into pcDNA3.1 plasmid. Restriction digestion of pGRA5 with Pst I enzyme showed correct in-sertion of GRA5 DNA into the plasmid. Sequence analysis revealed 100% homology with the published sequence of gra5. immunofluorescence and Western blotting analyses of HEK 293-T cells transfected with pGRA5 showed specific expression of GRA5. Immunogenicity of pGRA5 will be evaluated in mice
ABSTRACT
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 tToxoplasma 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 [GRA8], excluded from the signal peptide and C-terminal transmembrane domain, with a short fusion tag in Escherichia coli. [E.coli]. GRA8 was purified using an optimized single-step Iimmobilized Mmetal ion Aaffinity Cchromatography [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 GRA8 recognized a single antigen of T. gondii tachyzoite at the expected molecular weight of native GRA8. To diagnosis acute tToxoplasma infection in pregnant women, an indirect immunoglobulin M [IgM] 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
Subject(s)
Humans , Female , Antigens, Protozoan , Escherichia coli , Toxoplasmosis/diagnosis , Pregnancy , Immunocompromised Host , Gene Expression , Immunoglobulin M , Rabbits , Enzyme-Linked Immunosorbent AssayABSTRACT
Dense granule antigens [GRA antigens] of Toxoplasma gondii induce strong antibody response in humans and are considered as useful diagnostic antigens. Previous studies reported expression of amino terminal GRA8 protein in fusion with large tags such as glutathione-S-transferase. The present study aimed to produce soluble full length immunogenic GRA8 in bacteria. GRA8 complementary DNA [cDNA], encoding amino acids 24 to 258, was amplified from tachyzoites of RH strain and cloned in prokaryotic expression vector pET-28b[+]. Expression of recombinant GRA8 [rGRA8] was analyzed by SDS-PAGE. Antigenicity and immunogenicity of the protein were evaluated by Western-blotting. The cloned gene fragment exhibited complete similarity with the published sequence of GRA8 gene by sequence analysis. rGRAS was expressed in Escherichia coli infusion with a very small tag and the soluble protein was purified by immobilized metal ion affinity chromatography. In immunoblot, serum sample from a rabbit immunized with rGRAS recognized a single antigen of T. gondii tachyzoite at the expected molecular weight of native GRA8. Sera from acutely-infected pregnant women strongly reacted with rGRAS in Western-blotting, while sera from chronically-infected or T. gondii-negative women failed to recognize the protein. The full length soluble rGRAS was successfully produced in E. coli and shown to be a highly immunogenic protein. As a result it could be used in immunological as well as molecular biology experiments