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

Abstract

The 4D8 gene was recently discovered in Ixodes scapularis and identified as a tick protective antigen. Vaccination using recombinant 4D8 from I. scapularis showed a significant reduction against I. scapularis tick 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 of the presence of 4D8 and investigate its sequence homology in three Rhipicephalus tick species from Africa. The gene encoding this tick protective antigen in Rhipicephalus appendiculatus, Rhipicephalus decoloratus and Rhipicephalus microplus ticks was amplified, cloned, sequenced and expressed as a recombinant protein. The amino acid sequences between these three ticks species was found to be conserved with an identity of 96 to 98%. Recombinant 4D8 from the three tick species was expressed as a His-Tag fusion protein in Escherichia coli and the affinity-purified recombinant protein separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), then analyzed in immuno-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.

Key words: Ticks, vaccine, 4D8, conserved, antigen.