基金项目：高等学校博士学科点专项科研基金（20101106110021） 作者简介：时韦美（1990-），女，硕士，多基因疾病遗传易感性和药物遗传学研究 通信联系人：刘英（1961-），女，研究员，多基因疾病遗传易感性和药物遗传学研究；单基因疾病致病基因的定位克隆.
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。
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 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 HBeAg was examined by ELISA method. Result: The eukaryotic recombinant expression vector ADAR1 p110/p150-FLAG was successfully constructed. When HepG2.2.15 was transfected with ADAR1 p110/p150-FLAG, ADAR1 was notably upregulated and the supernatant HBsAg and HBeAg were significantly increased. Conclusion: The ADAR1 p110/p150-FLAG was sucessfully constructed and can be overexpressed well in HepG2.2.15. The upregulation of ADAR1 was associated with the secretion of HBsAg and HBeAg in HepG2.2.15, still further, promote the secretion of HBsAg and HBeAg.
Key words: ADAR1; overexpression; HepG2.2.15; HBsAg and HBeAg
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