Studies on RNase H of hepatitis B virus DNA polymerase to up-regulate the expression of cellular apoptosis susceptibility gene / 解放军医学杂志

ABSTRACT

Objective To study the activation effect of HBV RNase H protein on the transcription of cellular apoptosis susceptibility gene (CAS). Methods The promoter of DNA sequence of CAS gene was identified in GenBank by bioinformatics and amplified from HepG2 genome by PCR using sense (5′-GGTACCCGATTACATGTTGTACATGAAGG-3′) and antisence (5′-CTCGAGGCTGAGTTCCATTGCTATAG-3′) primers. As these primers contained Kpn I and Xho I recognition sites on their respective 5′-ends, the amplified DNA fragments were tested by sequencing and then subcloned into Kpn I/Xho I sites of pCAT3-Basic reporter vector by routine molecular biological methods. The reconstructed plasmid named pCAT3-CASp was identified by enzyme digestion of Kpn I/Xho I, in which the expression of chloramphenical acetyltransferase (CAT) was under the control of the promoter of CAS. The HepG2 cells were transfected by pCAT3-CASp, and then co-transfected by pCAT3-CASp and pcDNA3.1(-)-RH plasmids. At the same time, the empty pCAT3-basic and pCAT3-TXNRD1p were transfected (self-contructed by the authors) as controls. After 24h culturing, cells were collected and the expression of CAT activity was detected by ELISA according to the manufacturer′s protocol. Results The optical density of expression of CAT of pCAT3-CASp was 0.043 by ELISA, in contrast, the optical density of expression of pCAT3-Basic was 0.024. The expression of CAT in co-transfection of pCAT3-CASp and pcDNA3.1(-)-RH(0.065) was 1.5 times as higher as pCAT3-CASp plasmid (0.043), and 2.7 times as higher as pCAT3-Basic. Conclusions The CAS gene promoter identified in present study has transcription activity and HBV RNase H protein may activate the expression of CAS gene.