Biochemical properties of a purified protein in cystic fluid of <i>Taenia solium</i> metacestodes

, Cho, Seung Yull; , Kim, Suk Il; , Kang, Shin Yong; , Kong, Yoon

doi:1988.26.2.87

Korean J Parasito > Volume 26(2):1988 > Article

Original Article


Korean J Parasitol. 1988 Jun;26(2):87-94. English. Published online Mar 20, 1994. http://dx.doi.org/10.3347/kjp.1988.26.2.87
Copyright © 1988 by The Korean Society for Parasitology


Biochemical properties of a purified protein in cystic fluid of Taenia solium metacestodes
Seung-Yull Cho,Suk Il Kim,Shin Yong Kang and Yoon Kong
Department of Parasitology, College of Medicine, Chung-Ang University, Seoul 156-756, Korea.


Abstract
By affinity chromatography using a monoclonal antibody as ligand, Kim et al. (1986) purified a protein fraction in cystic fluid of Taenia solium metacestodes (CF). In this study, the biochemical properties of the purified protein were characterized. Discontinuous-polyacrylamide gel electrophoresis (disc-PAGE) of the protein at 4.5~10% separating gel concentration showed its molecular weight (MW) to be 150 kilodalton(kDa) in non-denatured state, while denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that it was composed of 3 different subunits with respective MW of 15, 10 and 7 kDa. Subunit of 7 kDa was shown to be linked to other subunits by disulfide bonds. Isoelectric point of the protein was pH 6.8. The protein was relatively heat-stable for immunologic analysis. These properties indicated that the protein, comprising about 70% of total content in CF, had similar biochemical characters with antigen B of Oriol et al.(1971) in hydatid cyst fluid (HF).

Figures


	Figs. 1a & 1b Disc-PAGE of CF of Taenia solium metacestodes(Fig. 1a) and purified A-antigen(Fig. 1b) at different separating gel concentrations. No. on the top of gels are; 1: 10% gel, 2: 9% gel, 3: 8% gel, 4: 7% gel, 5: 6%gel, 6: 5.5% gel, 7: 5% gel, and 8: 4.5% gel. A-E on left of Fig. 1a are component protein bands in CF.


	Fig. 2 Slope of "100×[log(Rf×100)]" against %gel concentrations in standard proteins and purified A-antigen.


	Fig. 3 Standard curve of molecular weight by minus slope in Fig. 2. Open circle ( ◦ ): standard proteins, closed circle ( • ): A-antigen (=band C in disc-PAGE), closed triangle (▴): component proteins in CF.


	Fig. 4 SDS-PAGE of CF, A- and U-antigen in 10~15% linear gradient gel. Mr: Molecular weight in kDa. Lane 1: standard proteins, lane 2: CF, lane 3: CF after boiling at 100℃ for 15 minutes, lane 4: A-antigen, lane 5: A-antigen, lane 7: U-antigen after boiling.

Fig. 5
SDS-PAGE/EITB of CF, A- and U-antigen using a polyclonal antibody (a patient serum). SDS-PAGE was done as in Fig. 4. Lanes 1 & 2: CF before (1) and after (2) boiling at 100℃ for 15 minutes, lanes 3 & 4: A-antigen before (3) and after (4) the boiling, lanes 5 & 6: U-antigen before (5) and after (6) boiling. Peroxidase conjugated antihuman IgG and 3,3'-diaminobenzidine/ H₂O₂ as substrate were used.

Fig. 6
SDS-PAGE of A-antigen at different conditions of sample treatment. M_r: molecular weight in kDa, lane 1: standard proteins, lane 2: A-antigen heated at 95℃ for 5 minutes with same amount of sample buffer containing 10% 2-mercaptoethanol, lane 3: A-antigen mixed with sample buffer containing 2-mercaptoethanol without heating, lane 4: A-antigen heated at 95℃ for 5 minutes with sample buffer without 2-mercaptoethanol, lane 5: A-antigen mixed with sample buffer without 2-mercaptoethanol and without heating.


	Fig. 7 IEF findings of CF and A-antigen. Lane 1:standard proteins with pH, lane 2: CF of T. solium metacestodes, lane 3: purified A-antigen (arrow head).

Tables


	Table 1 Change of antigenicity in 3 antigenic preparations(CF, A- and U-antigen) before and after boiling at 100℃ for 15 minutes. Absorbance of specific IgG antibody in 26 patients' sera were measured by ELISA (Cho et al., 1986)

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