Separation of protective antigens in a saline extract of Pasteurella multocida type 4 Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/8336h5164

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  • Protective antigens were separated from a 2.5% saline extract of Pasteurella multocida type 4 by chromatographic methods. The organism was grown for 18 to 20 hours at 37°C on an agar medium containing 5% heat-inactivated turkey serum. The bacterial growth was harvested in a 2.5% NaC1 solution and agitated for 1 hour at 56°C. The suspension was centrifuged, and the supernatant was dialyzed for 72 hours against 0.85% NaC1 solution (crude extract:CE). In gel filtration with a Sephadex G-200 column two protein peaks were obtained with CE. A protective antigen component was detected in the first protein peak (P-1) fraction, which was subsequently adsorbed onto DEAE-cellulose followed by elution by a linear gradient of NaCl. Two protein peaks were obtained. They uniformly contained an identical antigen, therefore were pooled (P-1'). P-1' was absorbed with anti-vaiole turkey serum antibodies by the use of an immunoadsorbent column to remove residual serum component originating from the growth medium. One protein peak (P-1") was obtained. In the gel diffusion and immunoelectrophoresis analyses P-1" produced one precipitin line against anti-CE serum. In sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) P-1" showed three major protein bands corresponding to the molecular weights 43,000, 31,000 and 24,500, and one carbohydrate band which did not correspond to any of the three protein bands. Fifty micrograms of the P-1" antigens protected turkeys against challenge exposure to the homologous strain.
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