Cloning and sequencing of a cDNA expressing a ribosomal P0 peptide from Culicoides nubeculosus (Diptera).

The research article presents a study conducted to identify the allergens causing Insect Bite Hypersensitivity (IBH), a type of allergic dermatitis affecting horses, primarily caused by bites from Culicoides spp. or sometimes Simulium spp. bugs. The team were able to identify a protein, named rCul n 1, which they believed could be the allergen causing IBH. However, their results showed no significant differences when comparing the immune response to rCul n 1 in normal horses and those affected with IBH.

Identification of Possible Allergens

• The research team screened a cDNA library that expressed proteins from Culicoides nubeculosus – the insect believed to cause IBH – using serum from a horse affected by IBH.
• They were able to identify one clone that showed an immunoreactive response.
• The sequence of the cDNA insert was determined, which showed it had a 600 base pair insert with an open reading frame that coded for a 78 amino acid long protein, which they named rCul n 1.

Protein Analysis and Expression

• The deduced amino acid sequence of rCul n 1 was analysed and revealed an identity of 67-78% with the C-terminal part of the 318 amino acid long ribosomal P0 protein from other Diptera (flies and mosquitoes).
• The 38 C-terminal amino acids also showed a 57% identity with a ribosomal protein from a type of fungus (Aspergillus fumigatus).
• The cDNA insert was then cloned and expressed as a tagged protein in bacteria (Escherichia coli) and purified.
• The recombinant rCul n 1 protein was found to bind to the antibody fraction used for screening the expression library.

Evaluation of the Protein’s Impact

• IgE and IgG levels against rCul n 1 in sera from 19 IBH-affected horses, 18 Swiss control horses, and 8 control horses from Iceland were determined using ELISA.
• No significant differences were detected between the three groups.
• rCul n 1 also did not induce the release of sulfidoleukotriene (sLT), a type of eicosanoid and primary mediator of the body’s inflammatory response.
• However, the release of sLT was observed when the horse leukocytes were exposed to whole body extracts of Culicoides.

Despite the initial finding that the rCul n 1 protein was immunologically reactive with sera from an IBH-affected horse, the subsequent investigation results suggest that it may not be the principal allergen causing the dermatitis. Additional allergens may be present in the Culicoides insect, triggering IBH. Further analysis is required for a comprehensive understanding of the cause of this equine allergic response.