RNA编辑酶ADAR1在HepG2215中过表达对其上清HBsAg和HBeAg的影响.docVIP

  RNA 编辑酶 ADAR1 在 HepG2.2.15 中过表 达对其上清 HBsAg 和 HBeAg 的影响# 时韦美，吴佳，伍晓盼，朱席琳，刘英** 5 （北京协和医学院基础学院生物化学与分子生物学系，北京 100005） 摘要：目的：克隆人 ADAR1 基因的两个转录本，构建携带 ADAR1 基因的重组真核表达载 体，瞬时转染 HepG2.2.15 细胞过表达，并探究其对 HepG2.2.15 上清 HBsAg 和 HBeAg 的影 响。方法：提取 Hela 细胞总 RNA 并反转录为 cDNA 作为模板，分段克隆，逐步连接，将 10 15 p110 和 p150 全长连接到 p3X-FLAG-CMV-14 表达载体，双酶切和测序鉴定重组载体；将重 组载体瞬时转染 HepG2.2.15 细胞系实现过表达后，提取细胞总 RNA 反转录为 cDNA，通过 实时荧光定量 PCR 检测确认过表达效果，上清除去细胞及碎片后 ELISA 法检测 HBsAg 和 HBeAg 水平。结果：成功构建重组载体 p110-FLAG/ p150-FLAG，瞬时转染 HepG2.2.15 细 胞过表达效果良好，过表达后上清中 HBsAg 和 HBeAg 均显著上升。结论： ADAR1 p110-FLAG/ p150-FLAG 重组载体构建成功，在 HepG2.2.15 细胞系过表达效果良好，过表达 ADAR1 p110-FLAG/ p150-FLAG 与 HepG2.2.15 细胞上清 HBsAg 和 HBeAg 的分泌相关，有 利于 HepG2.2.15 细胞分泌 HBsAg 和 HBeA。 关键词：ADAR1；过表达；HepG2.2.15；HbsAg 和 HBeAg 中图分类号：K394.3 20 RNA editing enzyme ADAR1 overexpressed in HepG2.2.15 and the effect on supernatant HBsAg and HBeAg SHI Weimei, WU Jia, WU Xiaopan, ZHU Xilin, LIU Ying (National Laboratory of Medical Molecular Biology,Institute of Basic Medical Sciendes,Chinese 25 30 35 40 Academy of Medical Sciences,School of Basic Medicine,Peking Union Medical College,Beijing 100005) Abstract: Objective: To clone the two trcanscripts of ADAR1(p110 and p150) , constuct its eukaryotic expression vector, and transiently transfect HepG2.2.15 cell line with the recombinant vector in order to explore the association of ADAR1 overexpresson and the supernatant level of HBsAg and HBeAg. Methods: The total RNA was extracted from Hela cells and then was reverse transcripted into cDNA. The full length of ADAR1 p110/p150 was cloned and ligated to p3X-FLAG-CMV-14 expression vector by fragment progessively. The recomibinant plasmid was confirmed by double enzyme digestion analysis and DNA sequencing. Transiently transfected HepG2.2.15 cell with ADAR1 p110/p150-FLAG to overexpress ADAR1, after that total RNA was extracted, cDNA was prepared by RT-PCR and real time quantitive PCR was performed to verify the overexpression effect. Finally, the level of HBsAg and