Cloning And Prokaryotic Expression Of Intestinal Trefoil Peptides TFF3Gene Of Swine

Intestinal trefoil peptide TFF3is a low molecular weight polypeptide, Which isalso known as intestinal trefoil factor (ITF), and is mainly expressed in intestine.It consistsof59amino acid residues,and molecular weight is approximately6.7Mr. Due to specialspatial structure, TFF3can promote intestinal mucosal cell proliferation, also can combinewith glycoprotein in the intestinal mucus and stabilize the intestinal mucus to maintain theintestinal mucus barrier, so TFF3can reduce the intestinal damage and promote intestinalrepair，which is of great significance. Therefore, it is a short peptide with potential medicinalvalue. The study of intestinal trefoil peptide’s function and application is of greatimportance，and will lay the foundation for further study of the TFF3’s physiological andpharmacological mechanism of action. Currently, TFF3gene is mainly obtained by geneticengineering methods. Studies have shown that, human galectin-1, one member of a largegalectin family, can function as a fusion partner to produce soluble folded recombinantswine TFF3in E.coli. In this test, we designed a new expression vector construct，pET28a-TFF3.Then pET28a-TFF3was transformed into E. coli BL21(DE3) pLysS andIsopropyl β-D-1-Thiogalactopyranoside(IPTG) was used to induce the expression ofrecombinant proteins.Lastly,we detected the proteins by the means of SDS-PAGE andWestern blot,and digested the proteins using Tev Protease to get intestinal trefoil peptideTFF3.The results showed that:1. The cloning of swine TFF3genePig intestines mRNA was extracted and reverse transcribed into cDNA. Then designprimer to amplificate TFF3gene. Its sequence length is180bp, and the coding proteininclude59amino acid residues.The molecular weight is about6.49kDa.2. The reform and construction of plasmid pET28a-TFF3The new plasmid derived from pET28a, whice contained the gene sequence of humangalectin-1, followed by the Tev protease cleavage site sequence, a6×His-coding sequence,and a multi-cloning site sequence where the cloned gene TFF3is inserted.3. The recombinant plasmid pET28a-TFF3was transformed into E.coli BL21(DE3) pLysScompetent cells and recombinant protein was induced to express The recombinant plasmid pET28a-TFF3was transformed into E. coli BL21(DE3)pLysS competent cells. By selecting different final concentrations of IPTG and differentinduction time for expression, the best conditions for protein expression was determined.Under the optimal induction conditions，after induced by IPTG, the expression of interestprotein was detected by SDS-PAGE electrophoresis.The results show that:the expressionprotein’s molecular weight is about25.3kD, and the most majority is soluble form. Mouseanti-6×His tag monoclonal antibody and HRP-labeled goat anti-mouse IgG secondaryantibodies were used in western blot to detect the expression protein, and the result showedthat the molecular weight of the expression protein was about25kDa, and it is the same asthe molecular weight of the target protein which we expected.4. The purification of recombinant proteins TFF3We purify the protein samples by using the His60Ni gravity columns.Then we doSDS-PAGE, and the result shows that we get a single purpose strip.So we get the purifiedfusion protein.5. Dialyze and condense the purified protein,and is digested by the TEV protease.ThenTricine-SDS-PAGEThe size of the expressed protein is approximately25.3kDa. After the TEV protease’sdigestion, the digestion product is precipitated with TCA and acetone precipitationmethod.Then we do the Tricine-SDS-PAGE electrophoresis. After the electrophoresis, thegel is stained by Coomassie brilliant blue.The result show that,there are two fragments,about16.94kDa and8.36kDa.