[Inhibition of Plasmodium yoelii circumsporozoite protein on the activation of nuclear transcription factor in hepatoma cells stimulated by TNF-alpha].

ABSTRACT

OBJECTIVE: To investigate on the effect of Plasmodium yoelii (BY265 strain) circumsporozoite protein (CSP) on the activation of nuclear transcription factor KB (NF-KB) in hepatoma cells (HepG2) stimulated by TNF-alpha. METHODS: Entire coding sequence of CSP was reverse transcribed and amplified by RT-PCR with sporozoite total RNA as template, then cloned into pFLAG-CMV8 for construction of the recombinant plasmid pFLAG-CMV8-CSP. Indirect immunofluorescence staining with rabbit anti-CSP was applied to verify the expression and distribution of FLAG-CSP fusion protein in HepG2. Three groups were established for the experiment group A with HepG2 transfected by pFLAG-CMV8 as negative control, group B with HepG2 transfected by pFLAG-CMV8 and stimulated by 100 ng/ml TNF-alpha, and group C with HepG2 transfected by pFLAG-CMV8-CSP and stimulated by 100 ng/ml TNF-alpha. Dual-luciferase assay and EMSA were performed to detect the nuclear translocation and activation of NF-kappaB, to observe if pFLAG-CMV8-CSP suppressed the activation of NF-KB in HepG2 stimulated by TNF-alpha. RESULT: The expression of pFLAG-CMV8-CSP was localized on cytoplasm of HepG2. The activity ratio of firefly luciferase to Renilla luciferase in group C (0.228 +/- 0.029) was significantly lower than both groups A (0.438 +/- 0.085) and B (0.571 +/- 0.030) (P < 0.05). EMSA showed that the band in group C was significantly weaker than group B. CONCLUSION: Plasmodium yoelii CSP localizes in the cytoplasm of HepG2 and inhibits the activation and nuclear translocation of NF-kappaB in HepG2 stimulated by TNF-alpha.