Cloning and expression of a gene encoding the flagellin of Clostridium chauvoei.
Clostridium chauvoei is a causative agent of blackleg and the major protective antigen of the organism is the flagellar protein. Using an Escherichia coli expression library of the C. chauvoei Okinawa strain, we isolated the fliC gene encoding the flagellin protein. DNA sequence analysis revealed an open reading frame of 413 amino acid residues with a calculated molecular mass of 43819Da. Comparison of the sequence with those of flagellins from other bacteria showed considerable homology in the N-terminal and C-terminal domains. The glutathione-S-transferase (GST)-flagellin fusion protein and the purified FliC protein after removing the GST part with thrombin reacted with both polyclonal antisera and the non-protective monoclonal antibody (Mab), Mo-114. However, the protective Mab, Mo-41, which may recognize its conformational epitope, failed to react with both the GST-flagellin fusion protein and the purified FliC. Furthermore, the GST-flagellin fusion protein and the purified FliC induced very little protective immunity in mice. These results suggested that a conformation-dependent epitope play an important role in the development of immunity against blackleg.
Kojima
A
A
National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries, 1-15-1, Tokura, Kokubunji, 185-8511, Tokyo, Japan. kojimaa@nval.go.jp
Uchida
I
I
Sekizaki
T
T
Sasaki
Y
Y
Ogikubo
Y
Y
Kijima
M
M
Tamura
Y
Y
D000818:Animals; D003001:Cloning, Molecular; D003013:Clostridium / Q000235:genetics*; D005408:Flagellin / Q000235:genetics* / Q000276:immunology; D015964:Gene Expression Regulation, Bacterial; D051379:Mice; D016366:Open Reading Frames; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000276:immunology; D017422:Sequence Analysis, DNA
D011994:Recombinant Proteins; D005408:Flagellin