ABSTRACT
Objective To investigate the immunodiagnostic valueofrecombinant granulocvte protein 5(rGRA5) protein in prokaryotic Toxoplasma gondii.Methods The PCR was used to ampliify GRA5 gene,then insert the target gene into the prokaryotic expression vector pET28a,and identified by double digestion and sequencing, then transfecte the correct targert gene into BL21 competent cells, SDS-PAGE and Western blot were used to detect the expression of the recombinant protein in BL21.ELISA was used to detect the serum of 100 cases of serologically Toxoplasma-positive individuals.Results The products of GRA5 gene of 363 bp in length was successfully amplified by PCR,the prokaryotic expression vector pET28a-GRA5 was constructed successfully, the result of double digestion showed that the insert fragment size was correct, and DNA sequencing results showed that the homology of GRA5 gene with GenBank was 100%.Also the expression of GRA5 protein was successfully detected in BL21 by Western blot(about 14 ku).ELISA method was used to detect 100 cases of patients with 73 cases showed positive results, the positive diagnosis rate was 73.0%.Among them, the positive detection rate of IgG positive samples was 72.5% in 40 cases,the positive detection rate of IgM positive samples was 53.3% in 30 cases,the positive detection rate of IgG and IgM positive samples was 93.3% in 30 cases.Conclusion The prokaryotic expression vector pET28a-GRA5 is constructed successfully, and the recombinant protein has potential for immunological diagnosis of toxoplasmosis.
ABSTRACT
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 μg/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.