Gene Cloning Of Endostatin From Rat And Its Expression In E.coli

OBJECTIVETo investigate gene cloning of endostatin from rat and its expression in Escherichia coliMETHODScDNA encoding rat endostatin was amplified from rat brain with RT-PCR and inserted into prokaryotic expression vector pThiohis, recombinant was identified by xbaI/kpnI double digestion, PCR and the nucleotide sequencing of the target gene. With recombinant pthiohis-Endo-TOP10 with cDNA encoding rat endostatin, fusional protein of rat endostatin was expressed by IPTG induced in fusional protein, and was purified to hemogeneity by affinity chromatography; The products of purified fusional protein digested by enzyme Enterokinasse(EK) were identified with SDS-PAGE and Western-bloting experiments,and the inflence of it on endothelial cell proliferation by MTT method assy.RESULTSThe size of the amplified endostatin gene fragment was in accord with the expected one matelly. And the gene sequence inserted into pThiohis was consistent with the known sequence after determination. The size of purified fusional protein was in accord with the that expected, the proliferation of endothelial cell was inhibited purified fusional protein also.CONCLUSIONThe recombinant of rat endostatin cDNA clone has been established successfully,and the fusional protein of rat endostatin with biological activity has been obtained. This study has provided a basis for further studies of endostatin's effects in vivo,and creates the conditions for final clinical trial.