Yong Sun1, Wei Wu, Yong Zhang, Shangjun Lv, Shiliang Wang, Xi Peng. 1. From the State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burn Research, Southwest Hospital, The Third Military Medical University, Chongqing, China.
Abstract
BACKGROUND: Intestinal trefoil factor (ITF), a member of the trefoil factor family, plays an important role in protecting the epithelial layer of the gastrointestinal tract from damage and repairing epithelium after injury. This study aims to produce ITF by Escherichia coli expression system and explore its role in the treatment of burn-induced acute gastric mucosal lesions. METHODS: Human ITF (hITF) gene encoding mature peptide was obtained by RT-PCR, and then inserted into the expression vector pET32a to construct the recombinant pET32a-hITF. After confirmation by gene sequencing, pET32a-hITF was transformed into E. coli Origami B(DE3), and TrxA-hITF fusion protein was expressed by conventional isopropyl-beta-d-thiogalactopyranoside induction in shake flask and analyzed with sodium dodecyl sulfate-polyacrylamide gel electorphoresis (SDS-PAGE) and Western-blot. Subsequently, TrxA-hITF was isolated by Nickel-nitrilotriacetic acid affinity chromatography, and ultrafiltration. Finally, a burn-induced rat gastric injury model was established, TrxA-hITF was administered orally and macroscopic and microscopic changes in gastric mucosa were observed. RESULTS: The correctness and integrity of hITF gene were identified by restriction digestion and gene sequencing. TrxA-hITF fusion protein was successfully expressed to 50 mg/L and its purity was above 95% after purification. SDS-PAGE and Western-blot analyses showed that the fusion protein presented as a single band with a molecular weight of 32 kDa, a little larger than the calculated value, 30 kDa. Lesion score and histologic examination revealed that TrxA-hITF alleviated rat gastric injury significantly. CONCLUSIONS: This study lays foundation for the commercial production of hITF and suggests a new way to treat burn-induced gastric lesions.
BACKGROUND:Intestinal trefoil factor (ITF), a member of the trefoil factor family, plays an important role in protecting the epithelial layer of the gastrointestinal tract from damage and repairing epithelium after injury. This study aims to produce ITF by Escherichia coli expression system and explore its role in the treatment of burn-induced acute gastric mucosal lesions. METHODS:HumanITF (hITF) gene encoding mature peptide was obtained by RT-PCR, and then inserted into the expression vector pET32a to construct the recombinant pET32a-hITF. After confirmation by gene sequencing, pET32a-hITF was transformed into E. coliOrigami B(DE3), and TrxA-hITF fusion protein was expressed by conventional isopropyl-beta-d-thiogalactopyranoside induction in shake flask and analyzed with sodium dodecyl sulfate-polyacrylamide gel electorphoresis (SDS-PAGE) and Western-blot. Subsequently, TrxA-hITF was isolated by Nickel-nitrilotriacetic acid affinity chromatography, and ultrafiltration. Finally, a burn-induced ratgastric injury model was established, TrxA-hITF was administered orally and macroscopic and microscopic changes in gastric mucosa were observed. RESULTS: The correctness and integrity of hITF gene were identified by restriction digestion and gene sequencing. TrxA-hITF fusion protein was successfully expressed to 50 mg/L and its purity was above 95% after purification. SDS-PAGE and Western-blot analyses showed that the fusion protein presented as a single band with a molecular weight of 32 kDa, a little larger than the calculated value, 30 kDa. Lesion score and histologic examination revealed that TrxA-hITF alleviated ratgastric injury significantly. CONCLUSIONS: This study lays foundation for the commercial production of hITF and suggests a new way to treat burn-induced gastric lesions.