Molecular cloning, sequencing and recombinant expression of a putative tick protective antigen from three ixodid ticks

Abstract

Ticks are arthropod vectors of pathogens impacting on human and animal health worldwide. Immunological control of pathogen vectors is an alternative control method that circumvents the drawbacks of chemical control. The 4D8 gene was recently discovered in Ixodes scapularis and identified as a tick protective antigen. Vaccination using recombinant 4D8 from 1. scapularis, showed a significant reduction against infestation in a sheep model. This protein is expressed in both salivary gland and gut tissues and is thought to be conserved in ixodid tick species. The objective of this study was to provide evidence for the presence of 4D8 and investigate its sequence homology in three Rhipicephalus tick species from Africa. The gene encoding this tick protective antigen in R. appendiculatus, R. decoloratus and R. microplus ticks was amplified, cloned, sequenced and expressed as a recombinant protein. The amino acid sequences were found to be conserved among these tick species with identity between 83% and 100%. Recombinant 4D8 from the 3 tick species was expressed as a His-Tag fusion protein in Escherichia coli. and the affmity-purified recombinant protein separated by SDS-PAGE then analyzed in irnmuno-blot analysis with anti-His- Tag antibody. A unique strong band of the predicted molecular weight of 17 kDa appeared suggesting presence of a protein corresponding to 4D8. These results confirm the presence of a 4D8 homologue in Rhipicephalus tick stocks from East Africa and further support the hypothesis that it is conserved in different tick species. This conservation among different tick species may suggest that it could potentially be an antigen in subunit vaccines for the control of multiple tick species.