A SIMPLIFIED METHOD FOR ISOLATING OUTER-MEMBRANE PROTEINS FROM PASTEURELLA-HAEMOLYTICA A1

The outer membrane of Pasteurella haemolytica Al (Ph-1) contains sever al major proteins which may play an important role in stimulating prot ective immunity. This report describes a method which was used to succ essfully isolate three Ph-1 outer membrane proteins (OMPs). The molecu lar weights of these OMPs are 31 kilodaltons (kD), 40 kD and 42 kD res pectively. This method involves two steps: first, a sarcosyl extractio n step was used to obtain a crude OMP prep; and secondly, these OMPs w ere separated by gel electrophoresis using a Bio-Rad 491 Prep Cell whi ch separates proteins based on their molecular weights. Isolation of t he proteins was demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunogenicity was then demonstrated by immunoblotting and enzyme-linked immuno-sorbent assays (ELISAs) wit h convalescent serum taken from calves with prior exposure to Ph-1. SD S-PAGE results show that single bands of each protein were obtained. I mmunoblots and ELISAs of these proteins show that the convalescent ant iserum used reacted with the 31 kD and 40 kD proteins, but did not rea ct with the 42 kD protein. This procedure provides for a simplified me thod to obtain highly purified OMPs from Ph-1 which may now be evaluat ed on an individual basis as to what role they may play in stimulating resistance to pasteurellosis. (C) 1997 Elsevier Science B.V.