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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
Mouse endostatin (ES) c DNA was amplified by RT-PCR using total RNA from Kunming mouse tissue as a template. Compared with the reported sequence (L 2 2 45 45 ), ES c DNA was obtained. There was a base difference between the ctg and the reported (gtg) at the 2 nd 78th base sequence, resulting in the encoded amino acid changed from Val reported in the literature to L eu. The ES c DNA has been accepted by Gen Bank and the accession number is Gen Bank AF2 5 7775. The ES c DNA was recombined into the eukaryotic expression vector p Sec Tag2 / Hygro B and transfected into COS-7 cells and detected by Western blotting. The results showed that the supernatant of the COS-7 supernatant of p Sec Tag2 - ES transcribed from 48 and 72 h had obvious ES expression bands. The 48 h transfection supernatant was used to act on human umbilical vein endothelial cells. The 3H incorporation experiment showed that the proliferation of endothelial cells was obvious. Suppressed. The results preliminary indicated that the ES c DNA cloned in this experiment was biologically active.