Preparation of recombinant glycoprotease of Pasteurella haemolytica A1 utilizing the Escherichia coli alpha-hemolysin secretion system.
Three murine monoclonal antibodies were prepared against the recombinant glycoprotease of Pasteurella haemolytica A1 expressed in Escherichia coli. These monoclonal antibodies were able to recognize the authentic glycoprotease from P. haemolytica A1 culture supernatant. A recombinant plasmid which contained most of the glycoprotease gene of P. haemolytica A1 fused with the secretion signal sequence from hlyA of the E. coli alpha-hemolysin determinant was constructed. This recombinant plasmid expressed a fusion protein (Gcp-F) which was secreted into the culture supernatant by E. coli cells when the alpha-hemolysin secretion functions HlyB and HlyD are supplied in trans. Gcp-F could be readily recovered from the supernatant free from other cellular materials and is suitable for use in vaccine trials and challenge experiments in animals.
Lo
R Y
RY
Department of Microbiology, University of Guelph, Ontario, Canada.
Watt
M A
MA
Gyroffy
S
S
Mellors
A
A
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D004731:Endotoxins / Q000032:analysis; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D006460:Hemolysin Proteins / Q000235:genetics; D016978:Mannheimia haemolytica / Q000201:enzymology* / Q000235:genetics; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D004731:Endotoxins; D006460:Hemolysin Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D008666:Metalloendopeptidases; C070738:O-sialoglycoprotein endopeptidase