Analysis of antigenic domain of GST fused major surface protein (p30) fragments of <i>Toxoplasma gondii</i>

, Nam, H W; , Im, K S; , Baek, E J; , Choi, W Y; , Cho, S Y

doi:1996.34.2.135

Korean J Parasito > Volume 34(2):1996 > Article

Original Article


Korean J Parasitol. 1996 Jun;34(2):135-141. English. Published online Jun 20, 1996. http://dx.doi.org/10.3347/kjp.1996.34.2.135
Copyright © 1996 by The Korean Society for Parasitology


Analysis of antigenic domain of GST fused major surface protein (p30) fragments of Toxoplasma gondii
H W Nam,^*K S Im,E J Baek,W Y Choi and S Y Cho
Department of Parasitology, Catholic University of Korea, School of Medicine, Seoul 137-701, Korea.

Received April 25, 1996; Accepted May 20, 1996.
Abstract
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.

Figures


	Fig. 1 Design of fragmentation of p30 intohydrohpilic and hydrohpobic moieties.


	Fig. 2 PCR-amplified p30 coding gene fragnents of T. gondii separated in 1.2% agarose gel. M: pBR328 DNA/Bgl I + Hinf I fragments; 1: PCR product of 1,011 bp for T37; 2: 762 bp for S28; 3 and 4: 516 bp for A19 and P19; 5: 246 bp for X9; 6: 270 bp for Y10; 7: 246 bp for Z9; and m: pBR322 DNA/Hae III fragments.

Fig. 3
Selected p30 DNA fragments for insertion into pGEX-4T-1 after EcoR I digestion.

M: lambda DNA/Hae III fragments; G: pGEX-4T-1 only; 1: pGEX-4T-1 with 1,011 bp for T37; 2: with 762 bp for S28; 3 and 4: with 516 bp for A19 and P19; 5: with 246pb for X9; 6: with 270 bp for Y10; 7: with 246 bp for Z9; m1: pBR328 DNA/Bgl I + Hinf I fragments; and m2: pBR322 DNA/Hae III fragments.


	Fig. 4 SDS-PAGE findings of expressed GST-p30 fusion proteins. W: wild JM105 cells; 1: GST-T37 of MW of 63 kDa; 2: GST-S28, 54 kDa; 3 and 4: GST-A19 and GST-P19, 45 kDa; 5: GST-X9, 35 Kda; 6: GST-Y10, 36 kAd; and 7: GST-Z9, 35 kDa, ad indicated with arrow heads. M: Molecular mass markers.


	Fig. 5 confirmation of GST fusion protein of p30 fragments by western blot. W: wild JM105 cells; and G: GST only. Others are described in Fig. 4 ad marked with asterisk(*).


	Fig. 6 Demonstration of rapid formation of inclusion body of GST-S28 fusion protein. S: supernatant after sonification and centrifugation in PBS and P: precipitaint. Numerals of x-axis ard induction time of hr at 30℃. GST_S28 fusion protein is indicated with right arrow head.


	Fig. 7 Western bolt analysis of RH tachyzoite extracts with patients sera of toxoplasmosis. NORMAL: reaction with normal human sera and PATIENT: reaction with positive human toxoplasmosis sera. Arrow head indicates reaction of sera to p30 of tachyzoites. *: selected serum for western blot in Fig. 8.


	Fig. 8 Western blot analysis of GST fusion proteins of p30 fragments using the patient serum. Refer to the legend of Fig. 5. GST fusion proteins of p30 fragments are marked with asterisk (*).

Tables


	Table 1 Primers designed for the amplification of p30 grne fragments

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