Role of the CPC sequence in the antioxidant activity of GcGAST protein in E.coli.

Gibberellic acid stimulated transcriptional protein from Gymnadenia conopsea (GcGAST) is a novel member of GA-induced cysteine-rich protein family, which shared 12 highly conserved cysteine residues with other members in C-terminal domain. In the present paper, the recombinant plasmid, as well as two mutants Serine-Proline-Cysteine (SPC) and Cysteine-Proline-Serine (CPS), were constructed to investigate for the first time the effects of the cysteines in Cysteine-Proline-Cysteine (CPC) sequence on the antioxidant activity of GcGAST protein. It was found that E.coli expressing wt GcGAST exhibited significant resistance against exogenous H(2)O(2). Similar phenomenon was observed for E.coli harboring SPC mutant. In contrast, the host cell overexpressing CPS mutant became more sensitive to H(2)O(2). Some studies on the level of inclusion body revealed that wt GcGAST and SPC mutant embedded in Inclusion bodies (IB) could effectively eliminate H(2)O(2), whereas the mutagenesis to Ser of the second Cys residue in CPC sequence gave rise to the compete loss of H(2)O(2)-eliminating ability. Fourier transform Infrared spectroscopy analysis indicated that the IB of CPS mutant contained more ß-sheet secondary structure than wt and SPC mutant. Non-reducing SDS-PAGE combined western-blotting analysis revealed that the disulfide bonds were important for the formation of IBs of wt GcGAST and SPC mutant, whereas non-reducing SDS-PAGE of resolubilized IBs showed that hydrophobic interaction favored the aggregation of IBs in CPS mutant. Taken together, these results suggested that GcGAST possessed antioxidant activity in the level of IB, which made some contribution to cellular resistance to H(2)O(2). More importantly, the second cysteine residue in CPC sequence was more essential for its antioxidant biological function.

[Dimo-thylidioctyl ammonium bromide-BCG polysaccharide nucleic acid adjuvant enhanced the immunogenicity of a Mycobacterium tuberculosis subunit vaccine].

OBJECTIVE: To investigate the activity of a novel adjuvant consisting of dimethyldioctyldecyl ammonium bromide (DDA) and BCG polysaccharide nucleic acid (BCG-PSN). METHODS: BCG-PSN was extracted by hot-phenol method, and combined with DDA and Mycobacterium tuberculosis fusion antigen AMM (Ag85B-MPT64(190-198)-Mtb8.4) to formulate the Mycobacterium tuberculosis subunit vaccine. Mice were immunized subcutaneously with a 2-week interval between the immunizations (0.2 ml/dose), and humoral and cell-mediated immunity were detected by ELISA and ELISPOT respectively. RESULTS: With the stimulation of Ag85B in vitro, the number of antigen specific IFN-gamma producing spleen lymphocytes were 222 +/- 79, 259 +/- 85, 230 +/- 64 per million respectively in the mice immunized with AMM + DDA + BCG-PSN, AMM + DDA, and BCG. Spleen lymphocytes in these 3 groups produced higher levels of IFN-gamma compared to the groups with the adjuvant of IFA or BCG-PSN alone or without adjuvant upon stimulation with Ag85B (t = 2.923-7.118, P < 0.05). Furthermore, the adjuvant consisting of DDA and BCG-PSN increased the ratio of Ig(2a)/IgG1 than DDA alone (0.125 vs. 0.025). Combined with AMM, the adjuvant DDA and the one consisting of DDA and BCG-PSN induced higher level of immunity than incomplete Freund's adjuvant (IFA), NaCl, and BCG-PSN alone. CONCLUSION: Mycobacterium tuberculosis subunit vaccine AMM + DDA + BCG-PSN induced a strong Th1-type immune response, and DDA + BCG-PSN, especially DDA promoted the immune response of the M. tuberculosis subunit vaccine in mice.