Purification of a Protective Antigen from a Saline Extract of Pasteurella multocida

Abstract

It has been shown previously that soluble material extracted from Pasteurella multocida P-1059 by a 2.5% NaCl solution protects turkeys from generalized septicemia at a subsequent challenge exposure to the organism. In the present study, a protective antigen was purified from the crude soluble material by chromatographic methods. Four protein peaks were obtained by gel filtration with Sephadex G-200. The protective antigen was detected only in the first peak fraction, which contained a substantial amount of carbohydrate. The peak 1 fraction was adsorbed onto DEAE-cellulose and eluted by a linear gradient of NaCl. Fractions corresponding to a single protein peak were pooled and passed through an immunoadsorbent column to remove any possible serum component originating from the growth medium. The purified antigen had a carbohydrate/protein ratio of 1.5 and formed a single precipitin line with rabbit antiserum against the crude material in gel diffusion and immunoelectrophoresis analyses. The antigen produced antibodies in rabbits and turkeys which formed a single precipitin line against the crude material. Upon sodium dodecyl sulfate-polyacryl-amide gel electrophoresis, the purified antigen showed three protein bands, corresponding to molecular weights of 44,000, 31,000, and 25,000, and one carbohydrate band. The carbohydrate band did not correspond to any of the three protein bands. Upon isoelectric focusing gel analysis, the purified antigen showed two bands (pI = 3.5 to 4.0 and 4.5 to 5.5), but the two bands were antigenically identical by isoelectric focusing crossed immunoelectrophoresis. The 50% protective dose of the purified antigen was between 10 and 50 μg of protein in trials where two doses were given at 14-day intervals to 10- to 20-week-old turkeys.