Expression, characterization, and serologic reactivity of recombinant surface antigen P22 of Toxoplasma gondii.
The utility of recombinant Toxoplasma gondii surface antigen P22 for the detection of specific T. gondii antibodies in human sera was evaluated. Polymerase chain reaction was used to produce a 438-bp fragment of the P22 gene; the fragment corresponded to the amino acids predicted to be in the processed, native antigen. The fragment was subcloned into pGEX-2T and was expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein. The fusion protein was purified in a soluble form and was found to be recognized by sera from infected individuals in immunoblots and an enzyme-linked immunosorbent assay. Immunoglobulin G antibodies in sera from 31 acutely infected patients in general reacted more strongly to the fusion protein than did those in sera from 31 patients with the chronic infection. None of the sera from a panel of 26 seronegative controls reacted with the fusion protein was separated from the GST partner by cleavage with thrombin, it retained its immunoreactivity and its electrophoretic mobility in polyacrylamide gels was found to be similar to that of native P22. By a modification of the published method for purification of the foreign polypeptide from the GST carrier, the recombinant P22 was readily purified to homogeneity by thrombin cleavage of the fusion protein while it was adsorbed to glutathione agarose.
Parmley
S F
SF
Department of Immunology and Infectious Diseases, Palo Alto Medical Foundation, California 94301.
Sgarlato
G D
GD
Mark
J
J
Prince
J B
JB
Remington
J S
JS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000953:Antigens, Protozoan / Q000032:analysis / Q000235:genetics / Q000276:immunology*; D000954:Antigens, Surface / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D006801:Humans; D007136:Immunoglobulins / Q000032:analysis; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology*; D014122:Toxoplasma / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; D000954:Antigens, Surface; D007136:Immunoglobulins; D011993:Recombinant Fusion Proteins