3 Quantitative real-time RT-PCR法によるPrevotella intermedia heat shock protein関連遺伝子の発現解析(第514回 大阪歯科学会例会)

抄録

Previously we used microarray analysis to demonstrate that expression of several heat shock protein genes were up-regulated in biofilm-forming Prevotella intermedia (P. intermedia) strain 17 as compared to the biofilm non-forming variant strain 17-2. We employed a real-time reverse transcription-polymerase chain reaction (RT-PCR) strategy to confirm the up-regulation of these genes in strain 17 recorded by a microarray. Total RNA was isolated from 6-, 12-, 18-, 24- and 30-hour cultures of strains 17, 17-2, and ATCC 25611 (a reference strain for P. intermedia). Real-time RT-PCR was performed according to the one-step RT-PCR protocol of iScript^<TM> One-Step RT-PCR Kit with SYBR^[○!R] Green (Bio-Rad Laboratories, Inc., Hercules, CA, USA). RT-PCR for 16 S rRNA was performed as an internal control. tThe target mRNA levels in strains 17, 17-2 and ATCC 25611 were defined and compared using Gene Expression Macro (Bio-Rad). The (Increased expression levels of heat shock protein genes, such as dnaK, grpE, dnaJ, groEL, groES, and clpB, were validated by real-time RT-PCR. Four out of six of the tested genes showed at least a 10-fold increase in average expression level in strain 17 as compared to that of strain 17-2 after a 12-hour culture period. Considering the up-regulation of the stress response genes in biofilm-forming strain 17, the biofilm formation in this organism might be associated with its stress response.