应用人工转录因子技术抑制乙型肝炎病毒复制  

Inhibition of HBV replication by constructing an artificial transcription factor

作　　者：李中堂[1] 罗伟[1] 付愚[1] 蒋清虎 刘济铭[1] 吴忠均[1] 

机构地区：[1]重庆医科大学附属第一医院肝胆外科,重庆400016

出　　处：《细胞与分子免疫学杂志》2015年第10期1322-1326,1331,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81171562)

摘　　要：目的设计与构建特异性结合乙型肝炎病毒增强子1(HBV Enh1)的锌指蛋白人工转录因子(ZFP-ATF),检测在Hep G2.2.15细胞内的表达、对细胞生长影响及观察其抑制HBV DNA复制与表达的作用。方法构建含结合结构域ZFP与抑制性效应结构域Krüppel相关盒(KRAB)的人工转录因子(ATF),进行相关修饰、优化后,将人工合成ATF核酸序列插入真核表达质粒载体pc DNA3.1(^+),测序鉴定其正确性,X-treme GENE HP转染Hep G2.2.15细胞,共聚焦显微镜下观察ATF蛋白表达情况,CCK-8法检测ATF对细胞生长的影响,ATF转染24、48、72 h后,采用实时定量PCR、ELISA、Western blot法检测对HBV DNA复制与表达的抑制作用。结果成功构建pc DNA3.1-ATF(nls-ZFP-KRAB-FLAG)真核表达载体,ATF在Hep G2.2.15细胞中能正常表达,且对细胞生长无明显的影响,ATF具有抑制HBV DNA复制与表达的作用,在转染72 h后抑制作用达最高,抑制率达68.0%。结论构建的ATF在Hep G2.2.15细胞中能够正常表达且无毒性作用,可特异性结合HBV Enh1靶序列并具有抑制HBV DNA复制与表达的作用。Objective To construct a zinc finger protein-artificial transcription factor （ZFP-ATF） specifically bound to hepatitis B virus （HBV） enhancer 1 （Enhl）, then detect its expression in HepG2.2.15 cells, observe its effect on cell growth and its role in inhibiting the replication and expression of HBV DNA. Methods The zinc finger protein （ZFP） specifically bound to HBV Enhl was fused with a Kroppel-associated box （KRAB） transcriptional repression domain to produce an artificial transcription factor （ATF）. After relative modification and optimization, the synthetic ATF nucleic acid sequence was inserted into the eukaryotic expression plasmid vector pcDNA3.1 （＋） and its sequence was identified. Then pcDNA3.1-ATF was transfected into HepG2.2.15 cells through X-tremeGENE HP. The expression of ATF was detected by confocal microscope. The impact of ATF on cell growth was measured by CCK-8 assay. The inhibitory effect on HBV DNA by ATF was assured by real-time quantitative PCR （qRT-PCR）, ELISA and Western blotting at 24, 48 and 72 hours after transfection. Results The pcDNA3. 1-ATF （nls-ZFP-KRAB-FLAG） eukaryotic expression plasmid vector was successfully constructed. ATF was expressed in HepG2.2. 15 cells as expected without obvious influence on cell growth. ATF repressed the replication and expression of HBV DNA, especially at 72 hours post-transfection when the inhibition rate was 68%. Conclusion The synthetic ATF can be expressed in HepG2.2. 15 cells normally without toxic effect on the cells and exert an inhibitory effect on the replication and expression of HBV DNA by specifically bound to HBV Enh1.

关 键 词：锌指蛋白 人工转录因子 乙型肝炎病毒 增强子 

分 类 号：R512.62[医药卫生—内科学] R392-33[医药卫生—临床医学]