병원성 가시아메바 superoxide dismutase의 분자생물학 및 면역학적 병원성 기전 연구

초록

Purpose : The purpose of this study is to characterize of several biochemical properties and elucidate a possible protective role of an iron-containing superoxide dismutase(Fe-SOD) in Acanthamoeba castellanii, involving the defense against the oxygen-dependent killing mechanism in host. The sensitivity of active oxygen derivative was investigated by RNAi method, compared knockdown Acanthamoeba with wild type Acanthamoeba in vitro. Methods : To clarify the molecular pathogenicity of AcFe-SOD involved in infection, Acanthamoeba with suppressed Fe-SOD via RNA interference(RNAi) was compounded. The RNAi transfection was done at 24h, 48h and 72h. Western blot analysis with anti-AcFeSOD mouse serum and real-time PCR were performed to confirm the knock down of Fe-SOD. Results : As results, The decrease of Fe-SOD signal in siRNA-transfected Acanthamoeba was observed in western blot analysis, while wild type Acanthamoeba exhibited no decrease. By reai-time PCR, the suppression of Fe-SOD expression was quantitatively confirmed. The expression was recovered at 72h later. AcFe-SOD was not considered as a essential gene through NTT assay. Conclusions : We demonstrated that Fe-SOD expression in A. castellanii cells significantly inhibited by transfection with siRNA. This RNAi method will contribute to study pathogenesis of Acanthamoeba infeciton.

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