Cloning, nucleotide sequence and expression of the gene encoding factor Xa inhibitor from the salivary glands of the tick, Ornithodoros savignyi.
The N-terminal sequence of the competitive and slow tight-binding factor Xa inhibitor (fXaI; Ki = 0.83 +/- 0.10 nM) isolated from the salivary glands of Ornithodoros savignyi ticks (Acari: Argasidae) was employed to design a degenerate gene-specific primer (GSP) for 3'-rapid amplification of cDNA ends (3'-RACE). The primer consisted of a sequence encoding for amino acid residues 5-11. A full-length gene was next constructed from the 3'-RACE product in a two-step PCR procedure and successfully expressed by the BAC-TO-BAC baculovirus expression system. The deduced amino acid sequence of the gene showed 46% identity and 78% homology to an fXaI (TAP) from Ornithodoros moubata. Recombinant fXaI (rfXaI) consists of 60 amino acid residues, has a molecular mass of approximately 7 kDa and inhibited fXa by approximately 91%. The availability of the rfXaI will aid further investigations of its potential for therapeutic applications and as vaccine against tick infestation. The authentic nucleotide sequence of the gene encoding tick fXaI furthermore enables studies at the genetic level and probing of other tick species for similar and related genes.
Joubert
A M
AM
Department of Biochemistry, University of Pretoria, South Africa.
Louw
A I
AI
Joubert
F
F
Neitz
A W
AW
D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants; D060829:Arthropod Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D065427:Factor Xa Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012469:Salivary Glands / Q000737:chemistry*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics*; D013987:Ticks / Q000235:genetics*
D000925:Anticoagulants; D060829:Arthropod Proteins; D018076:DNA, Complementary; D065427:Factor Xa Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C063967:tick anticoagulant peptide