持续释放人血管抑素的基因修饰化工程细胞hE/293细胞株的建立
人内皮抑素;真核表达载体;人胚胎肾细胞;hES细胞株;Westernblot,赵卉,潘静坤,罗芸,田磊,薛毅珑,赵卉,通讯作者:,ConstructionofgeneengineeredhE,293cellscontinuous
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赵卉, 潘静坤, 罗芸, 田磊, 薛毅珑, 中国人民解放军总医院老研所细胞室 北京市 100853
赵卉, 2004年石河子大学医学院硕士, 2005年至今中国人民解放军总医院博士在读, 主要从事生物型人工器官在临床的应用方面的研究.
通讯作者: 薛毅珑, 100853, 北京海淀区复兴路28号, 中国人民解放军总医院老研所细胞室. xueyl@plagh.com
电话: 010-66937914
收稿日期: 2007-02-09 接受日期: 2007-03-17
Construction of gene engineered hE/293 cells continuously releasing human endostatin
Hui Zhao, Jin-Kun Pan, Yun Luo, Lei Tian, Yi-Long Xue
Hui Zhao, Jin-Kun Pan, Yun Luo, Lei Tian, Yi-Long Xue,Institute of Geriatrics, General Hospital of Chinese PLA, Beijing 100853, China
Correspondence to:Yi-Long Xue, Institute of Geriatrics, General Hospital of Chinese PLA, 28 Fuxing Road, HaiDian District, Beijing 100853, China. xueyl@plagh.com
Received:2007-02-09 Accepted:2007-03-17
Abstract
AIM: To express the recombinant eukaryotic expression vector containing human endostatin gene in human embryonic kidney HEK293 cells, and construct a cell line continuously secreting human endostatin (hES).
METHODS: Human endostatin cDNA containing interleukin-2 (IL-2) secreting peptide was cloned into eukaryotic expression plasmid pSNA2 to construct the recombinant plasmid pSNA2/endostatin. The plasmid pSNA2/endostatin was transfected into HEK293 cells by cationic liposome. The positive cell clones were selected by G418 and then named hE/293 cells. The expression of endostatin protein was analyzed by Western blot. The release of biologically active endostatin was confirmed using assays of ECV304 proliferation and the angiogenesis experiment of chicken chorioallantoic membrane (CAM).
RESULTS: Enzyme digestion and sequence analysis confirmed that the eukaryotic expression vector pSNA2/endostatin had been successfully constructed. After G418 selection, 3 strains of cells stably expressing hES were obtained, named as hE/293 cells. Western blot showed that ES protein with a molecular weight of 20 kDa existed in the supernatant of hE/293 cells ......
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