Abstract
A λZAPII cDNA library ofEchinococcus granulosus larvae was expressed inEscherichia coli SURE cells. Screening of the library with a rabbit antiserum raised against total larval antigen yielded several immunoreactive clones. For analysis of the nucleotide sequence, in vivo excision into pBlueskript was carried out and the 3′ end of the cloned insert was sequenced. Three of these clones exhibited identical nucleotide sequences, suggesting expression of identical genes. The complete nucleotide sequence of the largest clone, EG36, with a 3.4-kb insert was determined, presenting an open reading frame of 2.59 kb. The predicted amino acid sequence showed 71.4% identity to theSchistosoma mansoni paramyosin and a significant homology to a 17 amino-acid peptide sequence from antigen B ofTaenia solium. From these data we conclude that EG36 is the paramyosin ofE. granulosus. For protein purification, the coding sequence of the cDNA was amplified by polymerase chain reaction and ligated in frame into the expression vector pGEX-3X. Affinity-chromatography-purified GST fusion protein was used to induce a polyclonal rabbit antiserum. Immunoblot analysis revealed the expression of a 97-kDa protein by theE. coli clone and that of a protein with a similar molecular weight in protoscolices fromE. granulosus andE. multilocularis as well as inE. granulosus cyst fluid. Immunofluorescence studies showed that EG36 was localized throughout the tegument ofE. granulosus andE. multilocularis larvae. Sera from patients suffering from echinococcosis, schistosomiasis, and neurocysticercosis reacted with the purified fusion protein when tested in an enzyme-linked immunosorbent assay.
Article PDF
Similar content being viewed by others
References
Alber T (1992) Structure of the leucine zipper. Curr Opin Genet Dev 2:205–210
Chirgwin JM, Przybyla AE, McDonald RJ, Rutte WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299
Cohen C (1982) Matching molecules in the catch mechanism. Proc Natl Acad Sci USA 79:3176–3182
Cohen C, Parry DAD (1990) α-Helical coiled coils and bundles: how to design an α-helical protein. Protein 7:1–15
Del Brutto OH, Sotelo J (1988) Neurocysticercosis: as update. Rev Infect Dis 10:1075–1087
Epstein HF, Miller DM, Oriz I, Berliner GC (1985) Myosin and paramyosin are organized about a newly identified core structure. J Cell Biol 100:904–915
Flanigan P, King CH, Lett RR, Nanduri J, Mahmoud AF (1989) Induction of resistance toSchistosoma mansoni infection in mice by purified parasite paramyosin. J Clin Invest 83:1010–1014
Flisser A, Woodhouse E, Larralde C (1980) Human cysticercosis: antigenes, antibodies and non responders. Clin Exp Immunol 39:27–37
Frosch PM, Frosch M, Pfister T, Schaad V, Bitter-Suermann D (1991) Cloning and characterization of an immunodominant major surface antigen ofEchinococcus multilocularis. Mol Biochem Parasitol 48:121–130
Garnier J, Osgnothorpe DJ, Robson B (1978) Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol 120:97–120
Gottstein B (1992) Molecular and immunological diagnosis of echinococcosis. Clin Microbiol Rev 5:248–261
Gottstein B, Müller N, Cryz SJ, Vogel M, Tanner I, Seebeck T (1990) Humoral and cellular immune response in mice and dogs induced by a recombinantEchinococcus multilocularis antigen produced by aSalmonella typhimurium vaccine strain. Parasite Immunol 12:163–174
Grandea AG, Tuyen LK, Asikin N, Davis TB, Phillipp M, Cohen C, McReynolds LA (1989) A λgt11 cDNA that encodesDirofilaria immittis paramyosin. Mol Biochem Parasitol 35:31–42
Guerra G, Flisser A, Canedo C, Laclette JP (1982) Biochemical and immunological characterization of antigen B purified from cysticerci ofTaenia solium. In: Flisser A, Willms K, Laclette JP, Larralde C, Ridaura C and Beltran F (eds) Cysticercosis: present state of knowledge and perspectives. Academic Press, New York, pp 437–451
Henikoff S (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351–359
James LJ, Pearce EJ (1988) The influence of an adjuvant on protective immunity by a non-living vaccine against schistosomiasis. J Immunol 140:2753–2759
Laclette JP, Merchant MT, Willms K (1987) Histological and ultrastructural localization of antigen B in the metacestode ofTaenia solium. J Parasitol 73:121–129
Laclette JP, Landa A, Arcos L, Willms K, Davis AE, Shoemaker CB (1991) Paramyosin is theSchistosoma mansoni (Trematoda) homologue of antigen B fromTaenia solium (Cestoda). Mol Biochem Parasitol 44:287–296
Laclette JP, Shoemaker CB, Richter D, Arcos A, Pante N, Cohen C, Bing D, Nicholson-Weller A (1992) Paramyosin inhibits complement C1. J Immunol 148:124–128
Lanar DE, Pearce EJ, James SL, Sher A (1986) Identification of paramyosin as schistosome antigen recognized by intradermally vaccinated mice. Science 234:593–596
Landschulz WH, Peter FJ, McKnight SL (1988) The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 240:1759–1764
Li BW, Chandrashekar R, Alvarez RM, Liftis F, Weil GJ (1991) Identification of paramyosin as a potential protective antigen againstBrugia malayi infection in jirds. Mol Biochem Parasitol 49:315–325
Matsumoto Y, Perry G, Levine RJC, Blanton R, Mahmoud AAF, Aikawa M (1988) Paramyosin and actin in schistosomal teguments. Nature 333:76–78
Pearce EJ, James SL, Dalton J, Barrall A, Ramos C, Strand M, Sher A (1986) Immunochemical characterization and purification of Sm-97, aSchistosoma mansoni antigen monospecifically recognized by antibodies from mice protectively immunized with a nonliving vaccine. J Immunol 137:3593–3600
Pearce EJ, James SL, Hieny S, Lanar DE, Sher A (1988) Induction of protective immunity againstSchistosoma mansoni by vaccination with schistosome paramyosin (Sm97), a non-surface parasite antigen. Proc Natl Acad Sci USA 85:5678–5682
Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi RG, Horn GT, Mulli KB, Ehrlich HA (1988) Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Short MJ, Fernandez JM, Sorge JA, Huse W (1988) λZap: a bacteriophage expression vector with in vivo excision properties. Nucleic Acids Res 16:7583–7600
Smith DB, Johnson KS (1988) Single step purification of polypeptides expressed inEscherichia coli as fusion proteins with glutathione S-transferase. Gene 67:31–40
Vinós J, Domingo A, Marco R, Cervera M (1991) Identification and characterization ofDrosophila melanogaster paramyosin. J Mol Biol 220:687–700
Wiest PM, Wisnewski AV, Johnson JH, Ramirez B, Kresina TF, Olds GR (1991) Screening of murine monoclonal antibodies against living schistosomula ofSchistosoma mansoni by radio-immunoassay. Int J Parasitol 21:449–454
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Mühlschlegel, F., Sygulla, L., Frosch, P. et al. Paramyosin ofEchinococcus granulosus: cDNA sequence and characterization of a tegumental antigen. Parasitol Res 79, 660–666 (1993). https://doi.org/10.1007/BF00932508
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00932508