CHARACTERIZATION OF IMMUNORELATED PEPTIDES TO PORCIDIN P1

Porcidin P1, an antimicrobial peptide purified from the granules of po rcine polymorphonuclear neutrophils (PMN) using ultrafiltration and re verse phase high performance liquid chromatography (RP-HPLC), was cova lently conjugated to BSA and used to generate monospecific polyclonal ascites. Antibodies raised against porcidin P1 were covalently coupled to an Affi-gel Hz affinity column and used for immuno-affinity chroma tography of peptides from porcine PMN cell extract. Eleven immunorelat ed peptides were eluted from the column from neutrophil cell extracts and purified to homogeneity by HPLC. The molecular weights of the immu norelated peptides were determined by mass spectral analysis and range d in size from 1.91 to 10.65 kDa. Of the 11 immunorelated peptides whi ch were bound to the affinity column, only six peptides were recognize d by the anti-porcidin antibodies after HPLC purification. Three immun oreactive peptides displayed potent antibacterial activity towards Sta phylococcus aureus and Escherichia coli, reducing viability by as much as 99.9% (> 3 log reduction in CFU) when 5 mu g/mL of each purified p eptide was used. The polyclonal monospecific antibodies also reacted w ith proteins from ovine and human PMN, illustrating possible structura l relationships between small antibacterial peptides from the differen t species.