Preparation Of Endostatin Protein And The Measurement Of Its Biologic Activity

AIM: To investigate the preparation of endostatin protein and its biologic activity on vascular endothelial cell.METHODS: pBlast-hEndostatin and pBlast-Mcs were identified by digesting with Nhe I and Sal I, by PCR reaction, by sequencing, and by alignments of PCR products with gene bank using NCBIBLAST software. The identified pBlast-hEndostatin as well as pBlast-Mcs were then purified with QIA-GEN Endofree plasmid maxi kit. The purified plasmids transfected human fibroblasts. The expression of endostatin was detected by RT-PCR,Western blot and immunohistochemistry. The endostatin protein produced by transfected fibroblasts was purified by ultrafiltration and affinity chromatography. The inhibitory action of endostatin on human umbilical vein endothelium was measured by MTT assay.RESULTS: pBlast-hEndostatin was found to contain human endostatin gene. Endostatin protein was produced by transfected fibroblasts. The inhibitory ratio of 2.5,5,10,20, 40,80mg/L endostatin on human umbilical vein endothelium for 48 hours were 8.5%,13.1%,27.7%,38.1%,56.7%,63.8% respectively. IC50 value was 34.5mg/L. No inhibitive action was found on fibroblasts.CONCLUSION: Endostatin protein can be produced by the transfected fibroblasts. The produced endostatin has inhibitory action on human umbilical vein endothelium and has no inhibitive action on fibroblasts.