Development and evaluation of recombinant GRA8 protein for the serodiagnosis of Toxoplasma gondii infection in goats

Abstract

Background: The development of sensitive and specific methods for detecting Toxoplasma gondii infection is critical for preventing and controlling toxoplasmosis in humans and other animals. Recently, various recombinant proteins have been used in serological tests for diagnosing toxoplasmosis. The production of these antigens is associated with live tachyzoites obtained from cell cultures or laboratory animals for genomic extraction to amplify target genes. Synthetic genes have gained a key role in recombinant protein production. For the first time, we demonstrated the production of the recombinant protein of the T. gondii dense granular antigen 8 (TgGRA8) gene based on commercial gene synthesis. Recombinant TgGRA8 plasmids were successfully expressed in an Escherichia coli system. The recombinant protein was affinity-purified and characterized via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Furthermore, the diagnostic potential of the recombinant protein was assessed using 306 field serum samples from goats via indirect enzyme-linked immunosorbent assay (iELISA) and the latex agglutination test (LAT).

Results: Western blotting using known positive serum samples from goats identified a single antigen at the expected molecular weight of TgGRA8 (27 kDa). iELISA illustrated that 15.40% of goat samples were positive for T. gondii-specific IgG antibodies. In addition, TgGRA8 provided high sensitivity and specificity, with significant concordance (91.83) and kappa values (0.69) compared with the results obtained using LAT.

Conclusion: Our findings highlight the production of a recombinant protein from a synthetic TgGRA8 gene and the ability to detect T. gondii infection in field samples. The sensitivity and specificity of TgGRA8 demonstrated that this protein could be a good serological marker for detecting specific IgG in goat sera.

Keywords: GRA8; Gene synthesis; Goat; Serodiagnosis; Toxoplasma gondii.

Fig. 1

Screening of the PCR product of the TgGRA8 gene. Lane M, 100-bp DNA ladder; Lane 1, 582-bp PCR product after digestion with NdeI and AgeI

Fig. 2

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis on the optimized expression of TgGRA8 in E. coli strain Rosetta (DE3), Coomassie blue stained. Lane M: protein molecular weight marker. Lane 1 to 3: pellet fractions of cells grown at 20 °C after induction with 1.0 mM IPTG (4,8 h and overnight). Lane 4 to 6: uninduced total cell lysate of E. coli strain Rosetta (DE3)-pET21a-TgGRA8 (4,8 h and overnight)

Fig. 3

a Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of expression of recombinant Toxoplasma gondii dense granular antigen 8 (TgGRA8) protein. Lane M, protein molecular weight marker; Lane 1, the soluble recombinant TgGRA8 protein was purified using anti-FLAG tag affinity resin. b Western blot analysis of purified recombinant TgGRA8. Lane M, protein molecular weight marker; Lane 1, the purified 27-kDa TgGRA8 protein was detected using an anti-FLAG tag antibody

Fig. 4

Western blot analysis. Purified proteins were separated via 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and then probed with known positive and negative goat sera. a Lane M, protein molecular weight marker; Lane 1, strong reactivity with known positive serum; b Lane M, protein molecular weight marker; Lane 1, result for negative serum

Fig. 5

Antibody response to Toxoplasma gondii in field sera from goats using recombinant T. gondii dense granular antigen 8 protein-based indirect enzyme-linked immunosorbent assays

Fig. 6

The complete nucleotide and amino acid sequence of T. gondii GRA8 (accession number: TGME49_054720). The expression region of GRA8 in this study is delineated by shading (encoding a 194-residual peptide)