Analysis of antigenic domain of GST fused major surface protein (p30) fragments of Toxoplasma gondii.
Antigenic domain of major surface protein (p30) of Toxoplasma gondii was analyzed after polymerase chain reaction (PCR) of its gene fragments. Hydrophilic or hydrophobic moiety of amino acid sequences were expressed as glutathione S-transferase (GST) fusion proteins. Fragments of p30 gene were as follows: T37, total p30 open reading frame (ORF); S28, total ORF excluding N-terminal signal sequence and C-terminal hydrophobic sequence: A19, N-terminal 2/3 parts of S28; P19, C-terminal 2/3 of S28; X9, N-terminal 1/3 part of S28; Y10, middle 1/3 of S28; and Z9, C-terminal 1/3 of S28, respectively. Primer of each fragment was synthesized to include clamp sequence of EcoR I restriction site. PCR amplified DNA was inserted into GST (26 kDa) expression vector, pGEX-4T-1 to transform into Escherichia coli (JM105 strain). GST fusion proteins were expressed with IPTG induction as 63, 54, 45, 45, 35, 36, and 35 kDa proteins measured by SDS-PAGE. Each fusion protein was confirmed with GST detection kit. Western blot analysis with the serum of a toxoplasmosis patient revealed antigenicity in proteins expressed by T37, S28, and A19 but not those by P18, X9, Y10, and Z9. Antigenicity of p30 seems to be located either in N-terminal 1/3 part in the presence of middle 1/3 part or in the oligopeptides between margins of the first and second 1/3 parts.
Nam
H W
HW
Department of Parasitology, Catholic University of Korea, School of Medicine, Seoul, Korea.
Im
K S
KS
Baek
E J
EJ
Choi
W Y
WY
Cho
S Y
SY
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000032:analysis* / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D016054:DNA, Protozoan / Q000032:analysis; D005982:Glutathione Transferase; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals; D015800:Protozoan Proteins / Q000032:analysis* / Q000235:genetics; D014122:Toxoplasma / Q000276:immunology*
D000953:Antigens, Protozoan; D016054:DNA, Protozoan; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D015800:Protozoan Proteins; D005982:Glutathione Transferase