[Effect of IFN-gamma and TNF-alpha on hepatitis B virus posttranscriptional regulatory elements].

ABSTRACT

OBJECTIVE: To explore the effect of IFN-gamma and TNF-alpha on hepatitis B virus posttranscriptional regulatory elements. METHODS: The objective eukaryotic expression vectors were constructed by molecular cloning and PCR-based site-directed mutagensis in vitro, and identification was performed using PCR and sequencing analysis. CAT assays were performed by using ELISA kit. RESULTS: The eukaryotic expression vectors containing HPRE segment and mutating point were constructed successfully as confirmed by sequencing analysis. The normalized CAT activity from recombinant pDM138 and control produced in transfected cells incubated in the absence of cytokine were 896 +/- 214 and 37 +/- 11 pg/ml, respectively. After incubation with IFN-gamma, TNF-alpha and IFN-gamma + TNF-alpha, the normalized CAT activity from recombinant pDM138 produced in transfected cells were 324 +/- 57, 396 +/- 82 and 175 +/- 36 pg/ml, respectively (t = 5.19,4.39, 6.68, P < 0.01). The effect of IFN-gamma and TNF-alpha were dose dependent over a range of 0 to 1,000 U/ml. CONCLUSION: The expression of HBV gene may be regulated by IFN-gamma and TNF-alpha through inhibiting the function of HPRE.