RESUMO
alpha-Acetolactate decarboxylase(alpha-ALDC)gene has been cloned from Baillus brevis using PCR amplification. The amplified 0.97 kb DNA fragment was confirmed to be known alpha- ALDC gene by DNA sequencing. The fragment was inserted into the vector pBV220 to construct an expression plasmid pBVYI. This recombinant plasmid over expressed alpha-ALDC in E. coli DH5alpha. The alpha-ALDC activity of recombinant bacterium was 10 000-fold higher than that of Bacillus brevis. After purification, the properties of the recombinant alpha-ALDC were studied. The activity of this enzyme could be stimulated by Mn(2+), Sn(2+) and inhibited by Zn(2+), Cd(2+), Fe(2+), Co(2+) and Cu(2+). Moreover, amino acid modifiers could inhibit differently its activity. The optimum pH of the enzyme reaction was 5.5.
RESUMO
Epitope LKVIRK on 47 kDa of heat shock protein (Hsp) 90 of Candida albicans, corresponding to residues 386-391 of the Hsp90, is recognized by patients recovering from invasive candidiasis. The efficacy of hybrid phage displaying epitope LKVIRK in the N-terminal region of the major coat protein (pVIII) in inducing anti-invasive candidiasis immune response was studied in C57BL/6 mice. Indirect phage-ELISA results demonstrated that the mice immunized with hybrid phage had significantly higher titers of epitope LKVIRK-specific serum IgG as compared to those immunized with heat-killed C. albicans (HK-CA). C57BL/6 mice immunized either with hybrid phage or with wild-type phage also developed significant levels of delayed-type hypersensitivity (DTH) response and splenocyte proliferation, as well as with HK-CA. In addition, high levels of IFN-gamma in the CD4(+) splenocytes from phage-immunized mice were detected as well during 1 week post-inoculation. Furthermore, mice immunized with hybrid phage acquired a resistance to systemic C. albicans infection as confirmed by fewer C. albicans cells in the kidneys, and had a longer lifespan compared to control groups following intravenous challenge with C. albicans. These results indicate that hybrid phage displaying epitope LKVIRK may serve as a potential vaccine conferring a resistance to systemic candidiasis.