ABSTRACT
Fuantai-03(FAT-03), isolated from the Dasyatis akajei, has a strong antiangiogenic activity. The recombinant Fuantai-03 (GST/rFAT-03) fusion protein can be obtained with the DNA recombination technology. In this study, expression conditions of GST/rFAT-03 were optimized by response surface experimental design method. The constructed engineering bacteria containing GST/rFAT-03 plasmid was induced by isopropy-beta-D-thiogalactosid (IPTG), the GST affinity column was used for isolation and purification, and then the effects of different culture time, IPTG concentration, induction temperature and induction time on the amount of soluble GST/rFAT-03 fusion protein were compared. The culture time for optimal expression was 6.13 h, IPTG concentration was 0.36 mmol/L, induction temperature was 19.71 degrees C, and induction time was 13.60 h. The amount of soluble GST/rFAT-03 fusion protein was 7.57 mg/L under above mentioned expression conditions. The results also showed that rFAT-03 significantly inhibited angiogenesis in chicken chorioallantoic membrane in a dose-dependent manner. Moreover, the soluble form of the target protein is useful for further work on purification and on studying its biological function.
Subject(s)
Animals , Angiogenesis Inhibitors , Genetics , Chickens , Chorioallantoic Membrane , Escherichia coli , Genetics , Metabolism , Fish Proteins , Genetics , Recombinant Fusion Proteins , Genetics , Skates, FishABSTRACT
Objective To investigate the effects of SDY-08 isolated from Dasyatis akajei on tis-sue repair. Methods MTT assay was performed to measure the effect of SDY-08 on the growth of ECV-304 cells. The effect of SDY-08 on angiogenesis was detected in the chick embryochorioallantoic membrane (CAM). Mouse wound model was applied to investigate the effect of SDY-08 on tissue repair. Immunohistochemical staining assay and Western blotting were adopted to examine the expression changes of vascular endothelial growth factor (VEGF), Bcl-2 and Bax in wound tissues in response to SDY-08. Results The proliferation rates of SDY-08 at final concentration of 80 μg/ml promoting ECV-304 cells were 28.1%, 115.6% and 81.4% respectively at 12, 24 and 36 hours. The induction rate of angiogene-sis of CAM by SDY-08 at concentration of 1.6 mg/ml was 72.1%. SDY-08 at 0.5 mg/ml markedly in-duced acceleration of wound healing in mouse model two days in advance. SDY-08 up-regulated either ex-pressions of VEGF in trauma group or that of Bcl-2 and VEGF of ECV-304 cells, but down-regulated ex-pression of Box. Conclusions SDY-08 can significantly promote angiogenesis and tissue repair, which is closely correlated with its effect of up-regulating expressions of VEGF and Bcl-2 as well with that of down-regulating expression of Box.