Purification and N-terminal amino acid sequencing of Echinococcus granulosus antigen 5.
Antigen 5, a major parasite-derived lipoprotein of unilocular hydatid (Echinococcus granulosus) cyst fluid, comprises subunits of 56-70 kDa which dissociate on disulphide bound reduction to two subunits of approximately 25 and 38 kDa on SDS-PAGE. The 38 kDa component is recognized as a potentially important diagnostic marker for cystic hydatid disease. Single dimensional SDS-PAGE, two dimensional (2-D) gel electrophoresis, modified '2-D' gel electrophoresis, immunoaffinity-purification and HLPC were employed to purify antigen 5. Subsequent N-terminal sequencing suggested that antigen 5 isoforms exist due to the identification of a single amino acid sequence with alternative residues at some positions. An 18 amino acid consensus N-terminal sequence was derived for antigen 5 as a result of comparing the sequences obtained by the different fractionation methods. No homology was revealed to any previously identified protein including putative antigen 5 amino acid sequences. A synthetic peptide, mimicking the N-terminal consensus sequence, did not bind with patient sera (confirmed positive to antigen 5) or an anti-antigen 5 MoAb. Protein sequences (16 residues compared) for the 38 kDa subunit from sheep and horse (representing different strains of E. granulosus) cyst fluids were very similar except for differences at three positions. 2-D and modified '2-D' gel electrophoresis, in combination with immunoblot analysis, indicated the presence of more than one protein in the 38 kDa region of SCF capable of binding the anti-antigen 5 MoAb.
Zhang
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Molecular Parasitology Unit, Australian Centre for International and Tropical Health and Nutrition, Queensland Institute of Medical Research, Brisbane, Australia.
McManus
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D000595:Amino Acid Sequence; D000818:Animals; D000947:Antigens, Helminth / Q000235:genetics* / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004443:Echinococcosis / Q000175:diagnosis / Q000662:veterinary; D004446:Echinococcus / Q000235:genetics* / Q000276:immunology*; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D015801:Helminth Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D006734:Horse Diseases / Q000175:diagnosis; D006736:Horses; D006801:Humans; D015151:Immunoblotting; D008074:Lipoproteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis
D000947:Antigens, Helminth; D015801:Helminth Proteins; D008074:Lipoproteins