Identification of an epitope of alpha-enolase (a candidate plasminogen receptor) by phage display.

摘要: Alpha-enolase is an ubiquitous cytoplasmic glycolytic enzyme which also exhibits cell surface mediated functions and a structural role in the lens of some species. An alpha-enolase related molecule (alpha-ERM) present on surfaces neutrophils, monocytes monocytoid cells has capacity to specifically bind plasminogen, suggesting that alpha-ERM may function as plasminogen receptor. We have generated monoclonal antibody (mAB), 9C12, against alpha-ERM. This mAB reacted with both purified human Western blotting linked immunosorbent assays (ELISA). 9C12 detected associated peripheral blood neutrophils U937 assessed by fluorescence activated sorting (FACS) analyses. In addition, recognized intracellular pool alpha-enolase/alpha-ERM permeabilized cells. A phage display approach was employed identify epitope 9C12. Random fragments 100-300 base pairs (bp), obtained from full length cDNA, were cloned into filamentous vector pComb3B, generate phage-displayed peptide library. Recombinant phages binding selected their DNA inserts characterized direct sequencing. All bound encoded common sequence DLDFKSPDDPSRYISP, spanning amino acids 257-272 alpha-enolase. located within external loop molecule. These data indicate this contains is, therefore, exposed surface, further share acid sequences.