Molecular Cloning and Characterisation of the Gene Encoding the Ascaris Allergen ABA-1

Spence, Heather Jane (1994) Molecular Cloning and Characterisation of the Gene Encoding the Ascaris Allergen ABA-1. PhD thesis, University of Glasgow.

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Abstract

A 14 kDa protein, named ABA-1, is the most abundant protein in the body fluid of adult Ascaris, and is released by the tissue-penetrating larval stages of the parasite during culture in vitro. Immunological work has shown that ABA-1 is an allergen, suggesting that the IgE-mediated hypersensitivity response to ABA-1 seen during Ascaris infection may contribute to the pathology of ascariasis. In order to further characterise ABA-1, a cDNA expression library was constructed using mRNA prepared from A. suum infective larvae. Screening with polyclonal rabbit antiserum raised to gel-excised ABA-1 protein led to the isolation of a cDNA clone with a 1255 bp insert. DNA sequencing of the entire insert revealed that it consists of two 399 bp repeats and one truncated repeat. The putative amino acid sequence derived from each of the repeats was identical to the partial N-terminal amino acid sequence of the native ABA-1 protein, which had previously been derived by direct peptide sequencing. These data can be taken to indicate that ABA-1 is translated as a polyprotein which is then processed into 14 kDa monomers. To allow characterisation of the genomic organisation of the aba-1 gene, two genomic aba-1 clones were isolated from an A.suum genomic library. Characterisation of these clones revealed that they both contain part of the aba-1 genomic gene and suggested that there are at least 15 repeats of 399 bp in the aba-1 gene; in addition, there appears to be a 4 kbp intron situated at the 3' end of the gene. Northern and Western blots were used to determine stage- and tissue-specificity of the expression of the aba-1 gene. These data revealed that ABA-1 is expressed constitutively throughout the life of the parasite and that it would appear to be expressed in all tissues of the adult worm, although it is only found as a polyprotein in the gut. The gene encoding the A. lumbricoides ABA-1 protein was cloned and characterised by the use of the Polymerase Chain Reaction. This revealed that, like the A. suum aba-1 gene, it is also consists of tandem repeats of approximately 400 bp, suggesting that it to is expressed initially as a polyprotein and then cleaved into 14 kDa monomers. Western blot analysis of the the native ABA-1 protein confirmed this suggestion.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Molecular biology, Genetics
Date of Award: 1994
Depositing User: Enlighten Team
Unique ID: glathesis:1994-75608
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 19:18
Last Modified: 19 Nov 2019 19:18
URI: http://theses.gla.ac.uk/id/eprint/75608

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