THE ISOLATION OF IMMUNOGENIC OUTER MEMBRANE PROTEINS FROM MANNHEIMIA HAEMOLYTICA SEROTYPE 1 BY USE OF SELECTIVE EXTRACTION AND IMMUNOAFFINITY CHROMATOGRAPHY

Authors: MCVICKER, JERRY - IA STATE UNIV., AMES, IA; Tabatabai, Louisa

Submitted to: American Journal of Veterinary Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/7/2002
Publication Date: 12/20/2002
Citation: MCVICKER, J.K., TABATABAI, L.B. THE ISOLATION OF IMMUNOGENIC OUTER MEMBRANE PROTEINS FROM MANNHEIMIA HAEMOLYTICA SEROTYPE 1 BY USE OF SELECTIVE EXTRACTION AND IMMUNOAFFINITY CHROMATOGRAPHY. AMERICAN JOURNAL OF VETERINARY RESEARCH. 2002. v. 63(12). p. 1634-1640.

Interpretive Summary: We prepared an antibody-bound column with immunoglobulins isolated from pooled calf serum from animals infected with Pasteurella (Mannheimia) haemolytica. The antibody column was used to isolate the major immuno- reactive proteins prepared from the outer membranes of P. haemolytica grown under iron-deficient conditions. This growth medium allows the up- regulation of important protein virulence factors and other iron-regulated proteins. Five proteins were isolated with molecular masses of 42, 30, 24, 20 and 15 kDa. The major protein, the 42 kDa protein, was further characterized by N-terminal sequencing. No known homologue was identified in the database. A major immunogenic carbohydrate fraction was co-purified with the proteins. Additional experiments need to be performed in order to determine whether or not the carbohydrate would hinder or enhance the development of vaccine preparations.

Technical Abstract: Objective - To use the calf's antibodies, produced in response to infection by Pasteurella (Mannheimia) haemolytica, for the identification and subsequent isolation of the dominant immunogenic antigens (from bacteria grown in iron-deficient media) by immunoaffinity chromatography. Sample population - Serum from 10 calves, actively infected with P. haemolytica, was used to prepare the immunoaffinity column. Procedure - An outer membrane protein (OMP) fraction was obtained from sonicated salt- extracted P. haemolytica cells by extraction with N-lauroy sarcosinate. An immunoaffinity column was prepared from the immunoglobulin fraction of serum from calves actively infected with P. haemolytica. The immuno- affinity column was used to isolate the dominant immunogenic proteins from the OMP fraction. The resultant immunogenic protein fraction was characterized by SDS-PAGE, immunoblotting, ELISA, and carbohydrate quantification. N-terminal sequencing was performed on the most prominent band (42 kDa). Results - Five immunogenic proteins with molecular masses of 42, 30, 24, and 15 kDa were isolated. The immunogenic protein fraction contained 512 carbohydrate. The immunoaffinity column capacity was 1 ug of immunogenic protein per milliliter of gel. The N-terminal sequence of the 42 kDa protein was Tyr-Gln-Thr-Tyr-Gln Ser-X-Lue-Gln, where X could not be identified. Conlcusion - Using this method, immunogenic proteins were isolated. A significant amount of carbohydrates were co-purified in the process.l Additional experimentation needs to be performed in order to determine whether the presence of the carbohydrate would hinder or enhance the development of vaccine preparations. This mehtod could potentially allow more rapid production of antigens for experiment vaccines.