重组人内皮抑素真核表达载体pCD-sEndo的构建和表达

Construction of recombinant eukaryotic expression vector for human endostatin and its expression in liver cancer cell line SMMC-7721

目的:构建表达人内皮抑素的重组真核表达载体,并将其转染人肝癌SMMC-7721细胞,观察其体外抑制血管生成的活性. 方法:将含有IL-2分泌肽的人endostatin全长cDNA插入真核表达载体pcDNA3.0产生重组质粒pCD—sEndo,利用阳离子脂质体介导将其体外转染人肝癌细胞SMMC- 7721细胞.G418筛选后得到阳性克隆,并被命名为SMMC/sEndo,采用Western—blot检测转染细胞上清中endostatin蛋白的表达,鸡胚绒毛尿囊膜(CAM)血管生成实验检测转染细胞上清中内皮抑素蛋白的生物活性. 结果:成功构建endostatin基因的真核表达质粒pCD—sEndo并经酶切和序列测定证实,通过阳离子脂质体转染SMMC-7721细胞,Western-blot显示转染细胞上清有人endostatin蛋白的表达,Mr 20 000,而转然空质粒对照组未检测到目的蛋白.CAM实验显示在给予内皮抑素蛋白的鸡胚绒毛膜血管稀疏,血管密度明显减少,并且随着蛋白量的增加,血管减少程度加强. 结论:重组endostatin真核表达质粒构建正确,转染SMMC-7721细胞后可有效表达具有生物学活性的人endostatin蛋白并能分泌到细胞外.

AIM： To construct the recombinant eukaryotic expression vector for human endostatin and express it in human liver cancer cell line SMMC-7721, and to observe its anti-angiogenesis activity in vitro. METHODS： Human endostatin cDNA containing interleukin-2 （IL-2） secreting peptide was cloned into eukaryotic expression plasmid pcDNA3.0 to construct recombinant plasmid pCD-sEndo. The plasmid pCD-sEndo was transfected intoSMMC-7721 cells by cationic liposome. The positive cell clones were selected by G418 and named SMMC/sEndo. The expression of endostatin protein was analyzed by Western-blot. The activity of endostatin protein in the supematant of SMMC/sEndo cells was explored by the angiogenesis experiment of chicken chorioallantoic membrane （CAM）. RESULTS： The eukaryotic expression vector pCD-sEndo was successfully constructed and was confirmed by enzyme digestion and sequence analysis. The endostatin protein was expressed in the supematant of SMMC/sEndo cells, about 20 ku in size. No expression of endostatin protein was found in the control cells. There were fewer blood vessels in the CAM treated with endostatin protein, and the blood vessel density markedly decreased. Furthermore, the density decreased with the increase of endostatin protein. CONCLUSION： The recombinant eukaryotic expression vector is correctly constructed. The human endostatin protein with the activity of anti-angiogenesis can be expressed and secreted in the supernatant of SMMC-7721 cells tranfected with pCD-sEndo.