关键词: 诱导表达, 人肠三叶因子, 大肠杆菌

Abstract:

Objective  To construct Escherichia coli (E.coli) expression vector of human intestinal trefoil factor (hITF), express recombinant hITF and analyze its biological activity. Methods  The hITF gene encoding mature peptide was amplified by RT-PCR, and then inserted into the expression vector pET32a. Recombinant plasmid pET32a-hITF was transformed into the Escherichia coli Origami B（DE3） and hITF was expressed by IPTG induction. hITF was purified by Ni-NTA affinity chromatography, and determined by SDS-PAGE, Western blotting, and N-terminal amino acid sequence analysis. Biological activity was assayed in an in vitro restitution model. Results  It was proved that the fragment amplified was inserted into the expression vector pET32a correctly by PCR and gene sequencing. SDS-PAGE analysis proved that the molecular weight of hITF was about 32kD, and Western blotting demonstrated that the expressive proteins had good antigenicity and specificity. The N-terminal 15 amino acid sequence was consistent with the theoretical value. In addition, hITF was proved to enhance migration activity. Conclusion  hITF Escherichia coli expression vector is successfully constructed and recombinant hITF is expressed.

Key words: Escherichia coli, Inductive expression, Human intestinal trefoil factor