[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 prolifetation. The results demonstrated primary that the cloned endostatin cDNA had biological activity.