Hemalin, a thrombin inhibitor isolated from a midgut cDNA library from the hard tick Haemaphysalis longicornis.
A full-length sequence of a thrombin inhibitor (designated as hemalin) from the midgut of parthenogenetic Haemaphysalis longicornis has been identified. Sequence analysis shows that this gene belongs to the Kunitz-type family, containing two Kunitz domains with high homology to boophilin, the thrombin inhibitor from Rhipicephalus (Boophilus) microplus. The recombinant protein expressed in insect cells delayed bovine plasma clotting time and inhibited both thrombin-induced fibrinogen clotting and platelet aggregation. A 20-kDa protein was detected from the midgut lysate with antiserum against recombinant hemalin. The gene is expressed at all stages of the tick except for the egg stage, and hemalin mRNA mainly in the midgut of the female adult tick. Real-time PCR analysis shows that this gene has a distinctly high expression level in the rapid bloodsucking period of the larvae, nymphs, and adults. Disruption of the hemalin gene by RNA interference led to a 2-day extension of the tick blood feeding period, and 27.7% of the RNA-treated ticks did not successfully complete the blood feeding. These findings indicate that the newly identified thrombin inhibitor from the midgut of H. longicornis might play an important role in tick blood feeding.
Liao
Min
M
Laboratory of Emerging Infectious Diseases, Department of Frontier Veterinary Medicine, Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan.
Zhou
Jinlin
J
Gong
Haiyan
H
Boldbaatar
Damdinsuren
D
Shirafuji
Rika
R
Battur
Banzragch
B
Nishikawa
Yoshifumi
Y
Fujisaki
Kozo
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004064:Digestive System / Q000737:chemistry; D015723:Gene Library; D015151:Immunoblotting; D036341:Intercellular Signaling Peptides and Proteins; D026863:Ixodidae / Q000235:genetics*; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects; D034622:RNA Interference; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013917:Thrombin / Q000037:antagonists & inhibitors*
D017931:DNA Primers; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; C046163:hemalin; D013917:Thrombin