Toxoplasma gondii: cloning, sequencing, expression, and antigenic characterization of ROP2, GRA5 and GRA7
Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase...
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| Veröffentlicht in: | Genetics and molecular research 2008-01, Vol.7 (2), p.305-313 |
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| creator | Igarashi, M Kano, F Tamekuni, K Kawasaki, P M Navarro, I T Vidotto, O Vidotto, M C Machado, R Z Garcia, J L |
| description | Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO vector which contains thioredoxin and polyhistidine tags at the C- and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 microg/ml growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. These proteins will be used for serodiagnosis and vaccination. |
| doi_str_mv | 10.4238/vol7-2gmr423 |
| format | Article |
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The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO vector which contains thioredoxin and polyhistidine tags at the C- and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 microg/ml growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. 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The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO vector which contains thioredoxin and polyhistidine tags at the C- and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 microg/ml growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. 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| subjects | Animals Antigens, Protozoan - genetics Antigens, Protozoan - immunology Antigens, Protozoan - metabolism Blotting, Western Cloning, Molecular Electrophoresis, Polyacrylamide Gel Escherichia coli Fluorescent Antibody Technique, Indirect Gene Expression Membrane Proteins - genetics Membrane Proteins - immunology Membrane Proteins - metabolism Protozoan Proteins - genetics Protozoan Proteins - immunology Protozoan Proteins - metabolism Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - immunology Recombinant Fusion Proteins - metabolism Toxoplasma - genetics Toxoplasma - immunology Toxoplasma - metabolism Toxoplasma gondii |
| title | Toxoplasma gondii: cloning, sequencing, expression, and antigenic characterization of ROP2, GRA5 and GRA7 |
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