Comparative Study of Hirudins and Encoding Genes in Hirudo nipponia and Hirudo tianjinensis

This study systematically compared the genetic characteristics and functional activities of hirudins and their encoding genes between Hirudo nipponia (Hnip1–3) and Hirudo tianjinensis (Htia1–3) through bioinformatics analysis, recombinant protein eukaryotic expression, and activity assays. The results revealed 42 nucleotide variation sites and 27 amino acid variation sites across both species. All six genes were expressed and significant pairwise differences between genes were detected within each species. All hirudins were identified as secretory proteins, with Hnip2, Hnip3, and Htia1 showing higher docking scores with thrombin. Four recombinant proteins (Hnip1, Hnip2, Htia1, and Htia2) exhibited antithrombin activity, with Hnip1 displaying the strongest activity. No significant differences were observed in the sequence variation, gene expression, physicochemical properties, predicted three-dimensional structures, or antithrombin activity of hirudins between the two leech species. This may stem from substantial heterogeneity in the genetic makeup and functional characteristics of distinct hirudins within each species, ultimately reducing the statistical power of these interspecific comparisons. Integrating gene expression profiles with recombinant protein activity assessments revealed that H. nipponia hirudins exhibit superior antithrombotic potency compared to those of H. tianjinensis. This study establishes a theoretical foundation for medicinal leech resource development and provides critical data for innovative antithrombotic drug discovery.