Screeing Of Interaction Proteins Of Human TFF3 By Yeast Two-hybrid

OBJECTIVE:To screen proteins that can interact with human trefoil factor 3 (hTFF3) by yest two-hybrid technique in human gastric cDNA library and identify the interaction by reverse yeast two-hybrid.METHODS:Human TFF3 gene was obtained by RT-PCR amplification and was cloned to yeast expression vector pDEST32(named pDEST-hTFF3 ) to construct a bait plasmid.The expression of hTFF3 in yeast was detected by western blot.Subsequently the bait plasmid and prey plasmid inserted with cDNA fragments of gastric cancer cell were transformed into yeast cells Mav203.Then the diploid yeasts were plated on synthetic dropout nutrient medium to screen positive clones and only those which could not only grow on SC-Leu-Trp-His +3AT and SC-Leu-Trp-Ura plate,but also activate X-gal were picked up to sequenced.The inserted sequences were analyzed with bioinformatics.To confirm the interaction between the candidate proteins and hTFF3,the bait plasmids expressing Aldo-Keto reductase 1C 1(AKR1C1),heterogeneous nuclear ribonucleoprotein AI(hnRNP Al),chaperonin containing TCPI-γ(CCT_γ) andβ-arresstin-l(ARRB 1) were recombined,and so did the prey plasmid expressing hTFF3.The recombined bait and prey plasmid~ were transformed into yeast Mav203 cell to confirm the interaction.RESOULTS:Obtain hTFF3 gene by RT-PCR and recombine the plasmid pDEST32-hTFF3 successfuly.By yeast two-hybrid technique,twenty-one positive clonies were screened out and twelve of them were sequenced successfully.Six known genes coding AKR1C 1,hnRNPA 1,CCTy,ARRB 1,immunoglobin heavy constant alpha2(IGH_α2);selenoprotein WI(SEPW1) and six genes with unknown function were screened.Reverse hybrid indicated that AKR1C1, hnRNPA 1,CCT_γand ARRB 1 might interact with hTFF3.CONCLUSION:Four human polypeptides/proteins including AKR1C1,hnRNP-A1, CCT_γand ARRB1 might interact with hTFF3 according yeast two-hybrid results.This result might provide new clues for the research of relationship between the genesis and progress of gastric cancer and hTFF3.