Expression and Characterisation of recombinant Hp-FAR-1 from the rodent gastrointestinal nematode, Heligmosomoides polygyrus
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Date
2005-09
Authors
Yahaya, Zary Shariman
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Abstract
The key hypothesis on which this thesis is based is that Hp-FAR-1 protein plays a major
role in the development of H polygyrus, a murine gastrointestinal nematode, in terms of
obtaining essentially retinol and lipid. It is believed also to have the capability of
interacting with the immune system of the host, either in helping the parasite to evade
the immune system or by causing the host immune system to be alerted about the
presence of the parasite.
Hp-FAR-1 is predicted to have no N-glycosylation sites but to have two types of
phosphorylation sites. It also has a compact globular protein structure, its predicted
tertiary protein structure containing 3 a - helixes. Phylogenetic analysis revealed that
Hp-FAR-1 belongs to the same group as Ancylostoma ceylanicum FAR proteins (AcFAR-
1 and Ac-FAR-2) and C. elegans FAR proteins (Ce-FAR-2 and Ce-FAR-6). Data
are presented confirming the successful cloning of Hp-FAR-1 protein with one amino
acid substitution and its expression in E. coli. The subsequent unsuccessful attempt to
express recombinant Hp-FAR-1 without any amino acid substitutions is also presented
and discussed. In ligand binding assay, recombinant Hp-FAR-1 was found to bind
DAUDA, all-trans retinol, cis -parinaric acid and also oleic acid by competition.
Immunoblot analysis demonstrated that the anti Hp-FAR-1 antibody bound strongly to
Hpolygyrus L4 and adult worm homogenates. No binding was observed to the ES
products of adult and L4 H polygyrus. Using Real Time PCR, it was shown that HpFAR-
1 protein is transcribed throughout the lifecycle, with the greatest quantity in the
L 1 and L4 stages.
The immuno-localisation experiment shows that Hp-FAR-1 is ubiquitously detected in
L3, L4 and adult transverse sections of H polygyrus, and immunoelectron microscopy
demonstrates expression in the cuticle and hypodermis of adult worms.
NIH mice vaccinated with rHP-FAR-1 showed strong antibody responses to the protein
and there was some indication of immunity against primary exposure to H polygyrus,
but further experimental work is needed to justify the conclusion.
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Keywords
Recombinant Hp-FAR-1 , Rodent gastrointestinal nematode , Heligmosomoides polygyrus