Efficient extracellular production of recombinant Escherichia coli heat-labile enterotoxin B subunit by using the expression/secretion system of Bacillus brevis and its mucosal immunoadjuvanticity.
A gene encoding the mature Escherichia coli heat-labile enterotoxin B subunit (LTB) was introduced in a vector pNU212 and expressed at high levels in Bacillus brevis HPD31. The maximum amount of recombinant LTB (rLTB) secreted into the modified 5PY medium containing erythromycin was about 350 mg l(-1) when cultivated at 30 degrees C for 8 days. The rLTB purified directly from the culture supernatant by using D-galactose immobilized agarose was identical to the native LTB with respect to the molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the amino terminal amino acid sequence. Western blot analysis with antiserum to cholera toxin B subunit (CTB) indicated that rLTB had cross-reactivity to native CTB and its GM1 binding ability was almost the same as that of the CTB. The rLTB predominantly showed the pentameric form when non-boiled samples were applied to SDS-PAGE. When rLTB was administered intranasally to mice with diphtheria toxoid (D(T)), it resulted in the substantial stimulation of D(T)-specific serum IgG antibody, and in the induction of moderate levels of D(T)-specific mucosal IgA antibody responses in the nasal cavities and in the lung, suggesting that purified rLTB acts as a promising immunoadjuvant on mucosal immunizations.
Kozuka
S
S
Department of Microbiology, Nagoya City University Medical School, Mizuho-ku, Nagoya, Japan. kozuka@med.nagoya-cu.ac.jp
Yasuda
Y
Y
Isaka
M
M
Masaki
N
N
Taniguchi
T
T
Matano
K
K
Moriyama
A
A
Ohkuma
K
K
Goto
N
N
Udaka
S
S
Tochikubo
K
K
D000281:Administration, Intranasal; D000818:Animals; D001407:Bacillus / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000008:administration & dosage / Q000096:biosynthesis* / Q000276:immunology* / Q000302:isolation & purification; D017077:Culture Media, Conditioned / Q000737:chemistry; D004168:Diphtheria Toxoid / Q000008:administration & dosage* / Q000276:immunology; D004768:Enterotoxins / Q000008:administration & dosage / Q000096:biosynthesis* / Q000276:immunology* / Q000302:isolation & purification; D029968:Escherichia coli Proteins; D005260:Female; D005822:Genetic Vectors; D018928:Immunity, Mucosal / Q000187:drug effects / Q000276:immunology; D007070:Immunoglobulin A / Q000097:blood; D007071:Immunoglobulin A, Secretory / Q000032:analysis / Q000096:biosynthesis; D007074:Immunoglobulin G / Q000097:blood; D007420:Intestine, Large / Q000378:metabolism; D007421:Intestine, Small / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D051379:Mice; D009297:Nasal Mucosa / Q000378:metabolism; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D014169:Transformation, Bacterial; D014621:Vagina / Q000378:metabolism
D001427:Bacterial Toxins; D017077:Culture Media, Conditioned; D004168:Diphtheria Toxoid; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D007070:Immunoglobulin A; D007071:Immunoglobulin A, Secretory; D007074:Immunoglobulin G; D011994:Recombinant Proteins; C027538:heat-labile enterotoxin, E coli