Construction and expression of trefoil factor 3 expressing vector for tandem affinity purification / 中国组织工程研究

ABSTRACT

BACKGROUND:As a novel growth factor, human intestinal trefoil factor (TFF3) can promote cel growth and migration, and increase cel resistance to apoptosis, and it plays a great role in maintaining the mucosa integrity, mucosa protection and repairing the injured mucosa, also it has been closely related to the tumor growth and progression. With the function of mucosa repair, and as the tumor biomarker, TFF3 has a promising clinical application, but its definite interacting protein and molecular mechanism is stil unclear. OBJECTIVE:To construct and express the TFF3 recombinant protein with the tandem tag of StrepII-6×His in the target cel s for further purifying its interaction protein in the native condition based on the tandem affinity purification technique. METHODS:The DNA sequence for the tag (StrepII-TEV-6×His) and TFF3 as template was got by chemical synthesis and PCR amplification respectively. They were fused by the restriction enzyme XbaI site, and the tag sequence was located at the C terminus of TFF3 protein. TFF3-tag fusion gene was cloned into the pCDNA3.0 using EcoRI+HindIII, thus the TFF3-tag expressing vector pTFF3-C-StH was constructed and transfected transiently into gastric cel AGS by lipofectin. The recombinant TFF3-tag protein was expressed and detected by western blot assay. RESULTS AND CONCLUSION:The expressing vector pTFF3-C-StH for tandem affinity purification was constructed successful y, and was confirmed further by restriction enzyme analysis and sequenced. The recombinant TFF3-C-StH protein of TFF3-tag was expressed in the AGS cel , and showed specific antigenicity by western blot assay. Thus this work provides experimental base for further purification of the TFF3 interacting proteins.