| PrefaceAngiogenesis is the process of formation of capillaries that sprout from existing blood vessels. Blood capillaries are primarily composed of endothelial cells. This complex process implies the presence of multiple controls, which can be turned on and off within a short period. The switch of angiogenesis phenotype depends upon the net balance between angiogenic stimulators and inhibitors, up - regulation of an-giogenic factors and down - regulation of angiogenic suppressors. Angiogenesis is the essential process for tumor growth and metastases, because tumors must develop an adequate vascular network to meet their increasing demands for nutrition and oxygen. The study of angiogenesis, and the promise of angiogenesis inhibition as a means of cancer therapy, has dramatically accelerated in the last several years. Within the family of angiogenesis inhibitors, angiostatin, an endothelial cell-specific inhibitor, has been demonstrated to be the most potent. Angiostatin, a multiple kringle (1-4) -containing fragment of plasminogen, is an effective natural inhibitor of tumor angiogenesis, which is a potent inhibitor of angiogenesis in vivo, and selectively inhibits endothelial cell proliferation and migration in vitro. However, the mechanism of action of angiostatin on endothelial cells is lessclear. Further structural and functional studies are hampered by a lack of soluble, correctly folded, biologically active protein in sufficient auantity. To overcome these limitations, we produced recombinant an-giostatin in a baculovirus expression system. Here we report the expression and analysis of recombinant human angiostatin produced by insect cells infected with recombinant angiostatin-baculovirus.MethodsGeneration of recombinant baculovires by co-transfection. The angiostatin baculovirus transfer vector was co-transfected with viral DNA into Sf9 cells according to the manufacturers protocol. To purify the recombinant virus, we used the plaque assay to screen the pure recombinant plaque and amplify it to generate P - 1 stock. After that we determined the presence of angiostatin gene in the putative recombinant virus with PCR analysis. Once the recombinant virus has been i-dentified, we amplified the P-1 stock to attain the large scale, high- titer viral stock in order to initiate expression studies. The recombinant angiostatin was produced from Spodoptera frugiperda 9 ( Sf9) insect cells infected by the high titer virus stock we have prepared. The time course for expression of recombinant protein was detected by SDS- PAGE and Western blot, which can determine the optimal multiplicity of infection (MOI) and the appropriate time of harvest for the protein. The recombinant protein was purfled by ProBond?purified system and its content was determined by bicinchoninic acid ( BCA) method. In vitro endothelial proliferation inhibiting activity of recombinant Angiostatin was examined with MTT method by using human umbilical vein endothelial cells ( HUVEC). In this test we could drawthe inhibition curve and calculated the IC50 To confirm its bio - activity further we do CAM vascular inhibition test in vivo.ResultsWe construct recombinant angiostatin baculovirus with a high virus titer(2 +108 pfu/ml) successfully. Recombinant angiostatin was effectively expressed in insect cells (Sf9) as 53 KD fusing protein and its expression level was about 90% of insect cellular total soluble proteins. The recombinant angiostatin protein could inhibit endothelial cell proliferation in vitro with IC50 value of 2. 3ug/ml and inhibited remarkably growth of vas in chorioallantoic membrane ( CAM ) .DiscussionsAngiogenesis, an essential process for tumor growth, is regulated by endothelial proliferation factors and their inhibitors such as angiostatin. Inhibition of tumor - induced angiogenesis may represent an effective cytostatic strategy. Angiostatin inhibits endothelial proliferation, angiogenesis, and tumor growth. , so it is very important to receive a great deal of soluble, correctly folde...
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