乙型肝炎病毒DNA多聚酶RNase H对细胞凋亡易感基因表达的上调作用

Studies on RNase H of hepatitis B virus DNA polymerase to up-regulate the expression of cellular apoptosis susceptibility gene

目的探讨乙型肝炎病毒DNA多聚酶(HBV DNA P)结构域蛋白RNase H对细胞凋亡易感基因(CAS)的转录激活作用。方法以HBV DNA P结构域蛋白RNase H的反式调节因子的基因表达谱芯片结果为基础,利用生物信息学技术确定CAS的启动子区域(CASp),扩增CASp并克隆至真核报告载体pCAT3-Basic中,构建pCAT3-CASp报告载体。分别以该质粒单独或与pcDNA3.1(-)-RH共转染肝癌细胞系HepG2细胞,ELISA法检测氯霉素乙酰转移酶(CAT)的表达活性,并以pCAT3-Basic空载体、pCAT3-TXNRD1p分别转染HepG2细胞作为阴性和阳性对照。结果pCAT3-CASp和pcDNA3.1(-)-RH瞬时共转染的HepG2细胞的CAT表达活性是pCAT3-CASp的1.5倍,是pCAT3-Basic空载体的2.7倍。结论本实验进一步验证了我室利用基因表达谱技术研究RNase H蛋白反式激活作用的结果。我室克隆的CAS启动子具有顺式激活下游基因的活性;HBV的RNase H蛋白具有对CAS的反式激活作用。

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 （5P-GGTACCCGATTACATGTTGTACATGAAC, G - 3＇） and antisence （5＇-CTCGAGGAGTTC- CATTGCTATAG-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 pCAT 3-Basic reporter vector by routine molecular biological methods. The reconstructed plasmid named pCAT 3-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 pCAT-CASp and pcDNA3. I（-）-RH plasmids. At the same time, the empty pCAT3 basic and pCAT3-TXNRDlp were transfected （self-contructed by the authors） as controls. After 24h culturing, cells were collected and the ex- pression 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 pCAT3-1Basic was 0. 024. The expression of CAT in co-transfection of pCAT3-CASp and pcDNA3. I（-）-RH（0. 065） was 1.5 times as higher as pCAT3-CASp plasmid （0. 043）, and 2.7 times as higher as pCAT3-Basic. Condusions The CAS gene promoter identified in present study has transcription activity and HBV RNase H protein may activate the expression of CAS gene.