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Molecular and immunological characterisation of tropomyosin from Anisakis pegreffii

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Abstract

Tropomyosin (TM) is a major allergen in shellfish, known to cross-react with mite, cockroach and/or some roundworm (nematode) TM. In this study, we aimed to express and purify TM from the parasitic nematode Anisakis pegreffii and also to characterise its cross-reactivity with TM from shellfish. A. pegreffii was isolated from the flathead tiger fish (Neoplatycephalus richardsoni) and characterised using single-strand conformation polymorphism (SSCP)-based sequencing of the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA. The recombinant tropomyosin (rTM) of A. pegreffii was expressed, purified and confirmed by immunohistochemistry, sequencing and LC-MS/MS analyses. Immunohistochemistry showed the muscle and the base layer of the third-stage larvae (L3) of A. pegreffii as the location of TM in A. pegreffii. The molecular relationship of TM of A. pegreffii with homologs from other nematodes and crustaceans was inferred from phylogenetic analysis. Immunogenicity of TM from A. pegreffii was tested by immunoblotting, which showed that rTM from A. pegreffii binds to IgE from sera of patients with allergy to crustaceans. Immunoblotting also showed that the anti-TM monoclonal antibody (MAb) did not recognise rTM from A. pegreffii. The rTM from A. pegreffii was, however, recognised by anti-TM polyclonal antibodies (PAbs) as well as anti-crustacean polyclonal antibodies (PAbs). The detection of specific serum IgE antibody against parasite TM has been proposed as a useful approach for the diagnosis of parasite-induced allergy. The findings of this study merit further exploration of the cross-reactive allergenic proteins of Anisakis for improved, future diagnosis of allergenic diseases.

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Acknowledgements

We thank Dr. Sandip Kamath for assisting with the immunoblots and Prof Robyn O’Hehir for donating serum samples.

Funding

Andreas Lopata received financial support via an ARC Future Fellowship for this work. Robin Gasser received support from the Australian Research Council (ARC), the National Health and Medical Research Council (NHMRC) of Australia, Yourgene Bioscience (Taiwan), Melbourne Water Corporation and The University of Melbourne.

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Correspondence to Peter M. Smooker.

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Informed, written consent was obtained from all study subjects, and experimental protocols were approved by The Alfred Hospital Research Ethics Committee (Project number 192/07) and the Monash University Human Ethics Committee (MUHREC CF08/0225).

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Asnoussi, A., Aibinu, I.E., Gasser, R.B. et al. Molecular and immunological characterisation of tropomyosin from Anisakis pegreffii . Parasitol Res 116, 3291–3301 (2017). https://doi.org/10.1007/s00436-017-5642-4

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  • DOI: https://doi.org/10.1007/s00436-017-5642-4

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