To obtain an anti-tumor peptide of Tumstatin and detect its biological activity,the nucleotide sequence encoding 185-203 amino acids(19peptide)of Tumstatin was synthesized and inserted into the fusion protein vector p...To obtain an anti-tumor peptide of Tumstatin and detect its biological activity,the nucleotide sequence encoding 185-203 amino acids(19peptide)of Tumstatin was synthesized and inserted into the fusion protein vector pTYB2.After identification by sequencing and restriction endonucle-ases,the recombined vector was transformed into BL-21(DE3)E.coli competent cells.Transformed E.coli BL-21(DE3)were induced by isopropyl-β-thiogalactopyranoside(IPTG),and then expressed.By 1,4-dithiothreitol(DTT)reduction,the soluble 19peptide was obtained from a chitin affinity chromatograph.The biological activity of 19peptide was determined by 3-[4,5-dimethylthiazol-2-y1]-2,5-dipheny-tetrazolium bromide(MTT)assay,cell growth curve,the effect of the ascitic fluid transfevent H22 hepatoma on mice and via histopathological slices.The purified 19peptide directly inhibited proliferation and migration of murine B16 melanoma cells,SMMC-7721hepatoma carcinoma cells and human umbilical vein endothelial cells(HUVEC).The tumor inhibition rate of mice ascitic fluid transfevent H22 hepatoma was 48.46%.Histopathological slices showed that it could promote tumor tissue necrosis and decrease the density of blood vessels.With higher anti-tumor activity,19peptide has the potential to become a novel,potent anti-tumor agent.展开更多
基金This paper was supported by the National Natural Science Foundation of China(Grant No.30472035)Heilongjiang Province Natural Science Foundation of China(No.TD2005-21)Heilongjiang Province Educational Science Foundation of China(No.11511104).
文摘To obtain an anti-tumor peptide of Tumstatin and detect its biological activity,the nucleotide sequence encoding 185-203 amino acids(19peptide)of Tumstatin was synthesized and inserted into the fusion protein vector pTYB2.After identification by sequencing and restriction endonucle-ases,the recombined vector was transformed into BL-21(DE3)E.coli competent cells.Transformed E.coli BL-21(DE3)were induced by isopropyl-β-thiogalactopyranoside(IPTG),and then expressed.By 1,4-dithiothreitol(DTT)reduction,the soluble 19peptide was obtained from a chitin affinity chromatograph.The biological activity of 19peptide was determined by 3-[4,5-dimethylthiazol-2-y1]-2,5-dipheny-tetrazolium bromide(MTT)assay,cell growth curve,the effect of the ascitic fluid transfevent H22 hepatoma on mice and via histopathological slices.The purified 19peptide directly inhibited proliferation and migration of murine B16 melanoma cells,SMMC-7721hepatoma carcinoma cells and human umbilical vein endothelial cells(HUVEC).The tumor inhibition rate of mice ascitic fluid transfevent H22 hepatoma was 48.46%.Histopathological slices showed that it could promote tumor tissue necrosis and decrease the density of blood vessels.With higher anti-tumor activity,19peptide has the potential to become a novel,potent anti-tumor agent.
