Production Of Intestinal Trefoil Factor In Pichia Pastoris And Preliminary Study Of Its Receptor On Intestinal Epithelial Cell

Background:Intestinal trefoil factor(ITF),a small,promoting mucosa proliferation polypeptide,is contained by one characteristic trefoil domain.Expression of ITF is closely related to that of mucin glycoproteins in diverse biological sources.ITF play an important role in both maintaining the barrier function of mucosal surfaces and facilitating healing after injury.Up to now,the research of ITF is extensive and profound,including tissue distribution,gene localization,gene recombination,amino acid sequence and spatial structure analysis as well as mass of function research.But the research progression of ITF receptor is slow-moving,there is or not ITF unique receptor in intestinal epithelial cell has no accepted argument.Some researchers deem ITF has no its own special receptor,it educes biological effects depend on epidermal growth factor receptor(EGFR),others presume there is ITF unique receptor in intestinal epithelial cell.But there is no credible direct evidence.Objective:To explore if ITF special receptor exists on the intestinal epithelial cell and it's disposition by radioligand binding assay and receptor mapping.Methods:1.Expression and purification of ITF:Successfully transformed Pichia pastoris strain X-33 which contained ITF gene encoding mature peptide were screened with Zeocin. Subsequently,ITF was constitutively expressed and purified by ammonium sulfate precipitation,Ni-NTA affinity chromatography and ultrafiltration,determined by SDS-PAGE and Western blot analysis.2.Radioligand binding assay of ITF receptor:Purified ITF was labeled by ¹²⁵I, determined the optimal temperature,reaction time,quantity of intestinal epithelial cell and concentration of unlabelled ITF.The binding assay was performed under this condition. Data from binding experiments were calculated by linear regression analysis of Scatchard plots,dissociation constant(K_D) value and maximum density of binding sites(B_max) of the cell was gained.Subsequently,analysis HT-29,5E6L,Hacat cell by the same method. Compare the K_D and B_max of different cells with addition of excess unlabeled EGF,ITF in the reaction,EGFR blocking peptide screened the binding sites of EGF,to prove ITF receptor presence on the intestinal epithelial cells.3.ITF receptor localization in intestinal epithelial cells:subcultured intestinal epithelial cells were digested by 0.5%protease at 37℃for 5 minutes,adjust the cells concentration of 5.0×10⁵ cells/ml and inoculate to 8×8mm coverslip.After cells adherence, washed the coverslip by PBS buffer 3 times for 3 minutes,and incubated with B-Phycoerythrin labeled ITF for various time at 4℃,At the end of incubations,cells were washed by PBS buffer 3 times for 3 minutes,put the cell coverslip on a slide and mounting by 90%glycerine,observed the disposition of ITF receptor on intestinal epithelial cells by laser confocal microscopy.Results:1.ITF was successfully expressed in Pichia pastoris,Tricine SDS-PAGE and Western blot analysis showed that ITF had much good antigenicity and specificity.The purity after purification was above 95%,the yield of ITF was about 50mg/L.2.Established optimal reaction conditions of radioligand binding assay:standard incubation temperature was 4℃,an incubation time of 30 min and cell concentration of 5.0×10⁵ cell/ml were used in radioligand binding assay experiments.A 4000pmol/ml unlabelled ITF concentration was used in competition experiments.Analysed IEC-6 cell, HT-29 cell,5E6L cell and Hacat cell by radioligand binding assay.According to comparison of each cells' K_D and B_max,ITF bind with IEC,HT-29 and 5E6L showed high affinity,and Hacat cells showed low affinity.3.Discovered that the B-Phycoerythrin labeled ITF gathered at membrane in the intestinal epithelial cells by laser confocal microscopy,as increased incubation time and concentration of BPE-ITF,B-Phycoerythrin labeled ITF transfered into cytoplasm and nucleus.Conclusion:1.ITF was successfully produced in Pichia pastoris expression system.The purity after purification above 95%and fit for radioligand binding analysis experiment. 2.Established optimal reaction conditions of radioligand binding assay,and demonstrated presence of ITF receptors on intestinal epithelial cells(IEC-6),colon carcinoma cells(HT-29) and small intestine epithelial cells(5E6L).Epidermic cells(Hacat) have no ITF specific receptor.3.EGF and ITF specific band with each receptor,competitive inhibition between EGF receptor and ITF receptor did not exist.It is indicated that there is ITF specific receptor on the intestinal epithelial cells.4.Successfully label ITF with B-Phycoerythrin,and certificate that there is ITF receptor on membrane of intestinal epithelial cells and labeled ITF could transfer into cytoplasm and nucleus.