分泌人内皮抑素基因工程细胞系的建立

Construction of Recombinant Eukaryotic Expression Vector for Human Endostatin and Its Expression in HEK293 Cells

目的建立稳定的基因工程细胞系。方法将含有白细胞介素-2(IL-2)分泌肽的人endostatin全长cDNA插入真核表达载体pSNA2.0,产生重组质粒pSNA2.0/hEndostatin,利用阳离子脂质体介导将其转染到人HEK293细胞中,G418筛选后得到阳性克隆hED/293,采用Western-blot检测转染细胞上清中endostatin蛋白的表达。结果hED/293细胞株培养上清中有endosta-tin蛋白的表达,分子量为20000。结论重组pSNA2.0/hEndostatin真核表达载体构建正确,转染HEK293细胞后可有效的表达人endostatin蛋白,并能分泌到细胞外。

Objective To construct a cell line with long time secreting endostatin. Methods Human endostatin cDNA containing interleukin-2 （IL-2） secreting peptide was cloned into eukaryotic expression plasmid pSNA2.0 to construct recombinant plasmid pSNA2.0/Endostatin. The plasmid pSNA2.0/Endostatin was transfected into HEK293 cells by cationic liposome. The positive cell clones were selected by G418 and named hED/293. The expression of endostatin protein was analyzed by Western-blot. Results Endostatin could be determined in the supernatant of hED/293 cells. Conclusion The recombinant eukaryotic expression vector is correctly constructed. The human endostatin protein can be expressed and secreted.