Abstract:

Objective To express, identify and characterize the serine protease inhibitory activity of Trichuris trichiura serine protease inhibitor 1（TtSerpin1）. Methods The nucleotide sequence encoding for TtSerpin1 (GenBank No.MF401634) was amplified from adult T. trichiura cDNA and ligated into pET32a-sumo vector to construct the recombinant plasmid pET32a-sumo/TtSerpin1. The positive recombinant plasmid was transferred into Escherichia coli BL21 (DE3) and expressed with IPTG induction. After denaturation, refolding, purification by Ni-NTA resin affinity chromatography and cleaving fusion tag by SUMO protease on the resin, a single stage chromogenic assay was used to detect the inhibitory activity of rTtSerpin1 against serine proteases. Results The recombinant plasmid pET32a-sumo/TtSerpin1 was constructed successfully. The fusion protein expressed in E. coli was insoluble and became aggregated as the inclusion body. The purified TtSerpin1 after refolding showed inhibitory activity against serine proteases. At a concentration of 1 000 nmol/L, rTtSerpin1 inhibited the enzymatic activity of human cathepsin G (100 nmol/L), human neutrophil elastase (10 nmol/L), human proteinase 3 (200 nmol/L), porcine pancreatic elastase (10 nmol/L) and bovine pancreatic α-chymotrypsin (1 nmol/L) by approximately 60.89%, 82.84%, 21.21%, 58.32%, and 96.98%, respectively, but it showed a weaker inhibitory activity against human trypsin (1 nmol/L), porcine trypsin (1 nmol/L) and human fibrinolytic enzyme (5 nmol/L). rTtSerpin1 inhibited human cathepsin G and neutrophil elastase with a Ki value of (949.80±91.51) and (242.70±53.41) nmol/L, respectively. Conclusion TtSerpin1 shows an inhibitory activity against multiple serine proteases.

Key words: Trichuris trichiura, Serine protease inhibitor, Prokaryotic expression, Cathepsin G, Neutrophil elastase, Proteinase 3