Molecular characterization of Rhipicephalus (Boophilus) microplus Bm86 homologue from Haemaphysalis longicornis ticks

One sequence in the EST database of a midgut cDNA library prepared from semi-engorged female Haemaphysalis longicornis ticks has been found to be a homologue of the Bm86 gene of Rhipicephalus (Boophilus) microplus ticks. The full-length sequence containing a 1785 bp open reading fragment (ORF) was obtained and designated as the Hl86 gene. The predicted amino acid sequence of the Hl86 gene shows a 37% identity to the Bm86 gene. Hl86 is predicted to be a GPI-anchored membrane-bound glycoprotein with a 19-amino acid signal sequence and a 22-amino acid hydrophobic region adjacent to the carboxyl terminus. The most important feature that Hl86 has in common with Bm86 is the repeated pattern of 6 cysteine residues forming epidermal growth factor (EGF)-like domains. RT-PCR analysis showed that Hl86 mRNA transcripts are expressed in all the life cycles of H. longicornis, and the expression was found in the midgut of the adult tick. The Hl86 was expressed in Escherichia coli as a gene10 fusion protein. Mouse anti-recombinant Hl86 serum recognized an 86 kDa protein band in the midgut lysate of semi-engorged ticks in Western blot analysis and showed a strong reaction on the luminal surface of midgut cells in an indirect immunofluorescent antibody test (IFAT). Silencing of the Hl86 gene by RNAi led to a significant reduction in the engorged tick body weight. This is the first report of cloning and characterization of the Bm86 homologue in different genera and species of ixodid and argasid ticks since Bm86 was first reported in 1989.