Cloning, Characterization, and Heterologous Expression of a Candidate Hirudin Gene from the Salivary Gland Transcriptome of Hirudo nipponia

Abstract Gene cloning and rapid amplification of cDNA ends (RACE) were used to determine the full-length cDNA sequence of a previously reported candidate hirudin gene (c16237_g1) in the salivary gland transcriptome of Hirudo nipponia Whitman. The 489-bp cDNA sequence (GenBank accession number: MN 116511) was predicted to encode an 83-aa polypeptide (including a 20-aa signal peptide sequence) and to possess several properties of the hirudin “core” motifs associated with binding to the thrombin catalytic pocket—including CLC and a GSNV region conservatively replaced by the chemically similar NSNL in H. nipponia. All six cysteine residues, presumably involved in three disulfide bonds, were also evolutionarily conserved. The putative protein’s aa sequence showed 62% (E-value = 2e-13), 54% (E-value = 6e-14), and 53% (E-value = 5e-13) similarity to HV3 (PA) (ALA22935.1), HV1 (VV) (ALA22934.1), and HV2 (ALA14576.1) sequences of H. medicinalis Linnaeus, respectively. A fusion expression vector (pPIC9K-Hirudin) was constructed to successfully produce GS115 transformants (GS115/pPIC9K-Hirudin). This confirmed candidate protein expression under inductive conditions, with antithrombin activity of 800 ATU/mL. These findings provide a basis for further elucidating the molecular anticoagulation mechanism of hirudin, helping fill China’s current commercial gap for engineered H. nipponia hirudin and meeting the considerable demand for hirudin-based clinical drugs, including products that target important cardiovascular and cerebrovascular diseases.