Preparation Of Recombinant Human Angiostatin And Study Its Specific Action On Vascular Endothelial Cell

Purpose: To prepare recombinant human angiostatin (rhAS) expressed in E.Coli to obtain high purified protein with biological activity,which will be suitable for use in clinical application in the future. Then study its specific action on vascular endothelial cell.Methods: pQE30 (Angiostatin) plasmid was transformed into E.Coli(M15) to obtain recombinant E.Coli (M15/pQE/AS) strain expressing rhAS. Fermentation was carried out under optimized condition to produce rhAS inclusion body(IB). rhAS IB was then solubilized and processed refolding to obtain purified protein. ELISA method for measuring the angiostatin protein content in serum was developed. The biological activity and pharmacodynamics of rhAS was evaluated on the model of human microvascular endothelial cell line (HMEC-1), chick chorioallantoic membranes(CAM) and nude mice bearing human pancreatic cancer. Study the specific action of rhAS and its receptor was performed on the model of HMEC-1, S180 tumor and Matrigel by flow cytometry, rhAS-Sepharose 4B affinity chromatography, 99Tcm-rhAS imaging, radioautography and histomorphology. Results: 1). Recombinant E.Coli (M15/pQE/AS) was induced maximally to produce rhAS by adding IPTG at concentration of 0.1mmol/L when E.Coli fermentation reaching 0.8-1.0 at A600nm, then continue cultivating for 6 hours. 2). The methods for solubilizing IB and rhAS protein refolding were developed and described as follows: IB was washed by turns with 0.2% sodium deoxycholate, ddH2O and 2 mmol/L Gu·HCl. Washed IB was dissolved with 8 mmol/L Gu·HCl containing 15 mmol/L DTT (1:10, v/v). Then the solution of Gu·HCl was diluted with Tris-HCl buffer (1:15, v/v). Finally the diluted solution above was dialyzed against the buffer containing 1mmol/L GSH and 0.1mmol/L GSSG. 3). rhAS protein has shown its biological activity effects in vitro and in vivo, resulting in inhibition and apoptosis in HMEC-1 cell line, inhibiting angiogenesis of CAM and growth of human pancreatic cancer. 4). An ELISA method for measuring angiostatin contents in human serum was developed and rhAS 5μg/ml in serum can be detected with specificity. 5). Obtained some rhAS binding proteins including ATP synthaseα/βsubunit and actin which was verified as...