| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 95 === Ag473, a vaccine candidate for the protection against Neisseria meningitidis infection, is a highly conserved lipoprotein of unknown function and predicted to form an extremely high degree of α-helical structure. The specific aims of this study were of two folds: to investigate the possible role of Ag473 plays in inflammation and to define the surface accessible region(s) of the Ag473 protein.
To achieve the first goal, the expression profiles of proinflammatory cytokines in the human cell lines nasopharyngeal carcinoma NPC076, neuroblastoma SK-N-SH, and glioblastoma G5T/VGH after being co-cultured with the wild type (WT) meningococci or the ag473-knockout mutant (MT473) were examined by RT-PCR. In NPC076 cell line, IL-1α, IL-1β, IL-6 and IL-8 were attenuated after coculturing with the WT for 24 h; similar effect was observed when co-cultured with MT473 in the presence of recombinant Ag473 (rAg473) but not with either one alone. In SK-N-SH, only expression of IL-1αand IL-1β was down regulated after co-culturing with the WT for 24 h. In G5T/VGH, the expression of IL-1α, IL-1β, IL-6, and TNF-α was stimulated by both WT and MT473 strains. However, a delayed cytokine expression in response to MT473 was observed compared to that of WT. Together, these results implicate that Ag473 may participate in the modulation of host immune response during infection.
To achieve the second goal, monoclonal antibodies (mAb) against the native Ag473 (4-7-3) and rAg473 (α-rAg473) were used as the primary antibody to probe the Western blots of E. coli lysates each containing an Ag473 variant deleted at different region. The results showed that the functional epitope of 4-7-3 lies in aa 48-60. Surprisingly, the fourα-rAg473 mAbs analyzed all recognized the aa 55-60 regions. Sequence analysis of the variable region genes cloned from the corresponding hybridomas confirmed that all antibodies analyzed were independent clones. These results indicate that aa 55-60 of Ag473 is an immunodominant region in both native and purified recombinant Ag473 proteins although subtle differences may exist.
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