摘要
目的 构建HBVX蛋白真核表达质粒并了解其对反式转录激活作用的影响。方法 用亚克隆方法构建HBVX蛋白真核表达质粒 ,以氯霉素乙酰转移酶转化率分析 (CAT试验 )揭示HBX蛋白的反式转录激活作用。结果 HBVX蛋白真核表达质粒转染HepaG2细胞后 ,以WesternBlot检测发现 2 μg、5 μg和 10 μg的 p5SGUTPLHBXDNA均能有效表达 ;CAT试验揭示 2 μg、5 μg、10μg的 p5SGUTPLHBXDNA对报道基因 pHE2×CAT的14 C -标记乙酰化氯霉素转化率分别为 (4 5 .5± 2 5 .9) %、(6 5 .6± 14.4) %和 (6 8.8± 19.7) %。
Objective To construct HBV X mammalian expression plasmid and establishment the method to detect transcriptional activity of HBV X protein. Methods HBV X mammalian expression plasmid pSG5UTPLHBX was constructed using subclone method. Choramphenicol Acetyltransferase Assay(CAT) was set up to detect the transcriptional transactivation of HBX protein in vitro. Results Transfected in HepaG2 cells, all 2 μg, 5 μg, 10 μg of pSG5UTPLHBX DNA were well expressed. When 2 μg, 5 μg, 10 μg of pSG5UTPLHBX DNA were transfected together with reporter gene pHEC 2×CAT in the HepaG2 cells, the CAT activities were (45.5 ±25.9)%, (65.6±14.4)%, and (68.8 ±19.7)%, respectively.Conclusions These results lay a fundation for further study of transcriptional transactivation of HBX protein.
出处
《中华传染病杂志》
CAS
CSCD
北大核心
2002年第4期222-224,共3页
Chinese Journal of Infectious Diseases
基金
浙江省自然科学基金资助项目 ( 4 910 10 )
关键词
HBV
X蛋白
真核表达
质粒
乙型肝炎
反式转录激活
HBV X protein
Mammalian expression
Trans-activation(genetics)
Transcription factors
Plasmids
