University of Guelph

Patricia E Shewen

Judith N Strommer

Raymond W H Lee

Doug Hodgins

Reggie Y Lo

Reggie Y C Lo

Bruce N Wilkie

Craig A Strathdee

The present invention provides nucleic acid sequences encoding a modified leukotoxin protein, wherein the modification comprises the removal of nucleic acid sequences encoding amino acids within hydrophobic transmembrane domains of full length leukotoxin protein, preferably from. The modified leukotoxin proteins are useful in vaccine compositions effective againstin animals.

Modified leukotoxin gene and protein

Modified leukotoxin protein

A serum-free vaccine effective against pneumonic pasteurellosis in cattle comprising a non-toxic leukotoxin specific for ruminant leukocytes is disclosed. The leukotoxin is prepared in a serum-free medium from a culture of Pasteurella haemolytica. The produced leukotoxin is harvested from the culture medium upon detecting a certain stage during the logarithmic phase of the cell growth to obtain the optimum concentration of produced cytotoxin in the serum-free medium. Cattle may be treated with the vaccine to develop anti-leukotoxic immunity to pneumonic pasterellosis.

Process for the production of vaccine for prevention of Pasteurella

Serum-free, cell-free vaccine effective against pneumonic pasteurellosis

The gene coding for Pasteurella haemolytica leukotoxin can be cloned in a plasmic expressed in Escherichia coli. The leukotoxin gene can be isolated from a clone bank of P. haemolytica. The clone bank is constructed by partial digestion of genomic DNA. The resultant 5 to 10 kilobase-pair fragments are ligated into plasmid vector pBR322. The resultant clones are screened for the production of P. haemolytica soluble antigens by a colony enzyme-linked immunosorbent assay blot method with a rabbit antiserum raised against the soluble antigens. The clones producing P. haemolytica soluble antigens are then analyzed for the production of the leukotoxin by a cytotoxicity assay with cells from a bovine leukemia-derived B-lymphocyte cell line as the target cells. Positive clones are identified, and subsequent restriction analysis of the recombinant plasmids shows the same insert DNA is cloned in the plasmid vector. The DNA sequence analysis of the insert DNA reveals regions coding for the leukotoxin.

Leukotoxin gene of pasteurella haemolytica

Growing community of inventors

Guelph, Canada

Patricia E Shewen