Cloning and expression of a 30 kDa surface antigen of Pasteurella haemolytica

Rebecca Craven, A. W. Confer, M. J. Gentry

Research output: Contribution to journalArticle

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Abstract

Pasteurella haemolytica biotype A serotype 1 is the principal ethiologic agent of bovine pneumonic pasteurellosis. A clear understanding of the pathogenesis of this disease and the mechanisms of resistance to it has been limited by a lack of information on the important antigens of the organisms. Using recombinant DNA techniques we have cloned a segment of DNA from P. haemolytica A1 that encodes three proteins of 28, 30, and 32 kDa. Two of these proteins, 30 and 28 kDa, react strongly on a Western blot with a bovine serum raised against live cell of P. haemolytica A1. The gene for the 30 kDa protein was localized to a 3.1 kbp EcoRI fragment, and expression of the 30 kDa protein was found to be independent of an E. coli promoter. The 30 kDa protein comigrated with a 30 kDa P. haemolytica protein that was susceptible to radioiodination and presumably exposed on the bacterial cell surface. The other principal radiolabeles P. haemolytica proteins were 100, 45, and 15 kDa. Antibodies against the 30 kDa protein, isolated from E. coli carrying the recombinant plasmid, recognized 30 kDa and 15 kDa proteins in P. haemolytica serotypes 1-15 and caused agglutination of whole P. haemolytica A1 cells. Cattle vaccinated with live P. haemolytica, P. haemolytica outer membrane proteins, or the cloned 30 kDa protein developed antibodies to the cloned 30 kDa protein as detected by Western blotting and densitometry. Sera were obtained from cattle vaccinated with live or killed P. haemolytica or saline and challenged with P. haemolytica. Those sera were evaluated for antibody responses to the cloned 30 kDa protein. High antibody responses to the 30 kDa protein significantly correlated (P<0.01) with resistance to challenge. From these studies it is concluded that the 30 kDa protein represents a surface antigen of P. haemolytica A1 that may be important in inducing immunity to P. haemolytica.

Original languageEnglish (US)
Pages (from-to)63-78
Number of pages16
JournalVeterinary Microbiology
Volume27
Issue number1
DOIs
StatePublished - Jan 1 1991

Fingerprint

Mannheimia haemolytica
surface antigens
Surface Antigens
Organism Cloning
molecular cloning
Proteins
proteins
antibodies
cattle
Antibody Formation
Pneumonic Pasteurellosis
Western Blotting
Western blotting
Serum
Mannheimia haemolytica serotype 1
pneumonic pasteurellosis
Escherichia coli
Disease Resistance
Recombinant DNA
Antibodies

All Science Journal Classification (ASJC) codes

  • Microbiology
  • veterinary(all)

Cite this

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title = "Cloning and expression of a 30 kDa surface antigen of Pasteurella haemolytica",
abstract = "Pasteurella haemolytica biotype A serotype 1 is the principal ethiologic agent of bovine pneumonic pasteurellosis. A clear understanding of the pathogenesis of this disease and the mechanisms of resistance to it has been limited by a lack of information on the important antigens of the organisms. Using recombinant DNA techniques we have cloned a segment of DNA from P. haemolytica A1 that encodes three proteins of 28, 30, and 32 kDa. Two of these proteins, 30 and 28 kDa, react strongly on a Western blot with a bovine serum raised against live cell of P. haemolytica A1. The gene for the 30 kDa protein was localized to a 3.1 kbp EcoRI fragment, and expression of the 30 kDa protein was found to be independent of an E. coli promoter. The 30 kDa protein comigrated with a 30 kDa P. haemolytica protein that was susceptible to radioiodination and presumably exposed on the bacterial cell surface. The other principal radiolabeles P. haemolytica proteins were 100, 45, and 15 kDa. Antibodies against the 30 kDa protein, isolated from E. coli carrying the recombinant plasmid, recognized 30 kDa and 15 kDa proteins in P. haemolytica serotypes 1-15 and caused agglutination of whole P. haemolytica A1 cells. Cattle vaccinated with live P. haemolytica, P. haemolytica outer membrane proteins, or the cloned 30 kDa protein developed antibodies to the cloned 30 kDa protein as detected by Western blotting and densitometry. Sera were obtained from cattle vaccinated with live or killed P. haemolytica or saline and challenged with P. haemolytica. Those sera were evaluated for antibody responses to the cloned 30 kDa protein. High antibody responses to the 30 kDa protein significantly correlated (P<0.01) with resistance to challenge. From these studies it is concluded that the 30 kDa protein represents a surface antigen of P. haemolytica A1 that may be important in inducing immunity to P. haemolytica.",
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Cloning and expression of a 30 kDa surface antigen of Pasteurella haemolytica. / Craven, Rebecca; Confer, A. W.; Gentry, M. J.

In: Veterinary Microbiology, Vol. 27, No. 1, 01.01.1991, p. 63-78.

Research output: Contribution to journalArticle

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