【摘要】 以中国昆明种小鼠组织的总 RNA为模板 ,用 RT- PCR方法扩增得到小鼠内皮抑制素 (ES)c DNA,与文献报道的序列 (L 2 2 5 45 )相比 ,ES c DNA测序结果 ctg在第 2 78碱基处与文献报道 (gtg)存在一个碱基差异 ,导致编码的氨基酸由文献报道的 Val变为 L eu。该 ES c DNA已被 Gen Bank接受 ,登录号为Gen Bank AF2 5 7775。将该 ES c DNA重组到真核表达载体 p Sec Tag2 / Hygro B上 ,转染 COS- 7细胞后 ,用Western blotting法检测。结果表明 48和 72 h的 p Sec Tag2 - ES转录 COS- 7上清有明显的 ES表达条带 ,取48h转染上清作用于人脐静脉内皮细胞 ,3H掺入实验显示内皮细胞的增殖明显受到抑制。该结果初步表明 ,本实验克隆的 ES c DNA有生物学活性更多还原

【Abstract】 The mouse endostatin cDNA was cloned by the total RNA of Chinese Kunming mouse liver as template with RT PCR.The results of sequencing showed one base pair difference.The ctg was replaced by gtg(L22545 in GenBank)at position 278 base pair,causing the encoded amino acid from reported Val to Leu in this experiment.This new endostatin cDNA was registered in GenBank with an accession number of AF257775.The recombinant eukaryotic expression plasmid pSecTag2 ES was then constructed and transfected into COS 7 cells for transient expression.The results of testing by Western blotting showed an expression fragment in supernatants of pSecTag2 ES transfected COS 7 cells at 48 and 72 hours of transfection.Cultured HUVECs were used to detect the biological effect of supernatants in pSecTag2 ES transfected COS 7 after 48 hours of transfection. 3H incorporation assay showed an obvious inhibition of endothelial cell proliferation.The results demonstrated primary that the cloned endostatin cDNA had biological activity.更多还原