小鼠内皮抑制素基因的克隆及生物学活性的初步分析

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）ｃＤＮＡ，与文献报道的序列（Ｌ２２５４５）相比，ＥＳｃＤＮＡ测序结果ｃｔｇ在第２７８碱基处与文献报道（ｇｔｇ）存在一个碱基差异，导致编码的迄基酸由文献报道的Ｖａｌ变为Ｌｅｕ。该ＥＳｃＤＮＡ已被ＧｅｎＢａｎｋ接受，登录号为ＧｅｎＢａｎｋＡＦ２５７７７５。将该ＥＳｃＤＮＡ重组到真核表达载达ｐＳｅｃＴａｇ２－ＥＳ转录Ｃ

Cloning of mouse endostatin and primary analysis on its biological activity

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.