Gene Cloning, Expression And Functional Characterization Of Antithrombin Gene From Dermacentor Silvarum

Antithrombin of ticks plays an important role in the blood sucking and blood meal digesting in the process of growth and development. In this study, an antithrombin-like gene sequence was cloned by screening the cDNA library of Dermacentor silvarum, named DS-AT. The full-length of the sequence is 616 bp.The ORF is 429 bp, and codes 142 amino acids. SignalP4.1 analysis online showed that it contains a signal peptide with 16 amino acid residues and a mature peptide with 126 amino acid residues. By domain analysis, DS-AT contains two BPTI-like domains and belongs to the Kunitz family. A BLAST search indicated that the protein coded by DS-AT gene shows 89% homology to boophilin, and a three-dimensional(3D) model was built on the basis of the published structure of boophilin. Real-time PCR analysis showed that this gene is expressed at all stages of the tick except for the egg and larva stage, and it is mainly expressed in the midgut and salivary glands of the female and male adult ticks. The recombinant proteins of DS-AT, with molecular weight of around 36 KDa, were produced by the method of prokaryotic expression, In anti-clotting activity and antibacterial activity assays, the results showed that the recombinant proteins of DS-AT have thrombin inhibition activity and some antibacterial activity.