Cloning and kinetics of expression of Brucella abortus heat shock proteins by baculovirus recombinants.
In an effort to develop genetically engineered Brucella abortus (BA) vaccines, the genes encoding heat shock proteins (HSPs) GroEL, GroES, and HtrA were cloned and expressed in the BAC-TO-BAC Baculovirus System, and the kinetics of protein expression were analyzed using various insect cell lines in suspension cultures, different cell densities in suspension cultures, multiplicities of infection and recombinant virus replication times. Trichoplusia ni cells expressed only BA HtrA, but Spodoptera frugiperda (Sf9) cells expressed all three recombinant proteins. The best GroEL expression was achieved by infecting 2x10(6) Sf9 cells/ml with an MOI 10 of recombinant virus and harvesting the cells after 96h of virus replication. GroES and HtrA were best expressed when infecting 2x10(6) Sf9 cells/ml with an MOI 1 of recombinant viruses and harvesting the cells after 120h of virus replications. Under these conditions BA recombinant HSPs were expressed as follows: GroEL at 16% of the total cellular proteins (105microg/ml concentration); GroES 2% (15.25microg/ml); and HtrA 8% (84.48microg/ml). This is the first report of cloning and expression of BA genes in the baculovirus system.
Bae
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Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0342, USA.
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D000818:Animals; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D001428:Bacterial Vaccines / Q000276:immunology; D015139:Blotting, Southern / Q000662:veterinary; D015153:Blotting, Western / Q000662:veterinary; D002003:Brucella abortus / Q000235:genetics / Q000276:immunology*; D002007:Brucellosis, Bovine / Q000276:immunology* / Q000517:prevention & control; D002417:Cattle; D002460:Cell Line; D018835:Chaperonin 10 / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D018834:Chaperonin 60 / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D005260:Female; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D033903:Periplasmic Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D018411:Spodoptera
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D018835:Chaperonin 10; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D033903:Periplasmic Proteins; D011993:Recombinant Fusion Proteins; C061682:DegP protease; D012697:Serine Endopeptidases