<正>Objective:To construct a Pichia pastoris （P. pastoris） expression vector of human intestinal trefoil factor （hITF） and study its expression and purification procedures. Methods:hITF gene encoding mature peptide was modified with a polyhistidine tag sequence at the N-terminal

and then inserted into the P. pastoris expression vector pGAPZαA at the downstream of theα-mating factor signal. After gene sequencing

the recombinant pGAPZαA-hITF was transformed into the P. pastoris strain X-33 with lithium chloride. rhITF was induced to constitutively express in shake flask

and then analyzed with Tricine SDS-PAGE and Western blotting. The obtained rhITF was isolated from the cultured supernatants by ammonium sulfate precipitation

Ni-NTA affinity chromatography

and ultrafiltration. Results:The correctness and integrity of rhITF were identified by restriction digestion and gene sequencing. rhITF was successfully expressed to 50 mg/L as a secretive protein. After purification

the purity was above 95%. Tricine SDS-PAGE and Western-blot analysis showed that rhITF presented as a single band with a molecular weight of 10 kDa

a little larger than 7 879 Da as assayed by mass spectrometry analysis. Conclusion: hITF P. pastoris expression vector is successfully constructed and rhITF is expressed in P. pastoris at commercially relevant level. This research lays foundation for the further functional studying of hITF.