基金項目：高等學校博士學科點專項科研基金（20101106110021） 作者簡介：時韋美（1990-），女，碩士，多基因疾病遺傳易感性和藥物遺傳學研究 通信聯系人：劉英（1961-），女，研究員，多基因疾病遺傳易感性和藥物遺傳學研究；單基因疾病致病基因的定位克隆.
摘要：目的：克隆人ADAR1基因的兩個轉錄本，構建攜帶ADAR1基因的重組真核表達載體，瞬時轉染HepG2.2.15細胞過表達，并探究其對HepG2.2.15上清HBsAg和HBeAg的影響。方法：提取Hela細胞總RNA并反轉錄為cDNA作為模板，分段克隆，逐步連接，將p110和p150全長連接到p3X-FLAG-CMV-14表達載體，雙酶切和測序鑒定重組載體；將重組載體瞬時轉染HepG2.2.15細胞系實現過表達后，提取細胞總RNA反轉錄為cDNA，通過實時熒光定量PCR檢測確認過表達效果，上清除去細胞及碎片后ELISA法檢測HBsAg和HBeAg水平。結果：成功構建重組載體p110-FLAG/ p150-FLAG，瞬時轉染HepG2.2.15細胞過表達效果良好，過表達后上清中HBsAg和HBeAg均顯著上升。結論：ADAR1 p110-FLAG/ p150-FLAG重組載體構建成功，在HepG2.2.15細胞系過表達效果良好，過表達 ADAR1 p110-FLAG/ p150-FLAG與HepG2.2.15細胞上清HBsAg和HBeAg的分泌相關，有利于HepG2.2.15細胞分泌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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