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 subun it (LTB) was introduced in a vector pNU212 and expressed at high levels in Bacillus brevis HPD31. The maximum amount of recombinant LTB (rLTB) secrete d 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 F rom the culture supernatant by using D-galactose immobilized agarose was id entical to the native LTB with respect to the molecular weight determined b y sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the amino terminal amino acid sequence. Western blot analysis with antiser um to cholera toxin B subunit (CTB) indicated that rLTB had cross-reactivit y 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 t o mice with diphtheria toroid (D-T) it resulted in the substantial stimulat ion of D-T-specific serum IgG antibody, and in the induction of moderate le vels of D-T-specific mucosal IgA antibody responses in the nasal cavities a nd in the lung, suggesting that purified rLTB acts as a promising immunoadj uvant on mucosal immunizations. (C) 2000 Elsevier Science Ltd. All rights r eserved.