Expression and activity determination of human angiostatin (k1-3) in culture cells and larvae of silkworm.
Angiostatin (k1-3) gene was inserted into Bombyx mori baculovirus transfer vector pBacPAK8 and cotransfected with lineared DNA of Bm-BacPAK6 virus into BmN cells. The homologous recombination occurred inside the cells, and the recombinant virus BacPAK-angiostatin was expressed, as identified by DNA dot blotting. The BmN cells and fifth instars were infected by the recombinant virus BacPAK-angiostatin; expression product was run in the SDS-PAGE, and its immunoreactivity was determined by using ELISA and Western blotting. The bio-activity of the protein product was determined by using human umbilical vein endothelial cells ( ECV304 ) proliferation test in vitro and by using CAM vascular inhibition test in vivo. The expression activity achieved the highest point at the 72nd hour in BmN cells (22 u / 2x10(6) cells) and at 144th hour in larvae (159 u/ml). At the concentration of 2.5 u/ml, angiostatin induced apoptosis of endothelial cells in 24 h and also inhibited angiogenesis in CAM.
Wang
Ying-Fei
YF
Institute of Biochemistry, Zhejiang University, Hangzhou 310029, China. summer.wang@yeah.net
Jin
Yong-Feng
YF
Zhang
Yao-Zhou
YZ
D000482:Allantois / Q000098:blood supply / Q000187:drug effects; D020533:Angiogenesis Inhibitors / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D043168:Angiostatins; D000818:Animals; D015153:Blotting, Western; D012831:Bombyx / Q000166:cytology / Q000235:genetics*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002642:Chick Embryo; D002823:Chorion / Q000098:blood supply / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000648:ultrastructure; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007814:Larva / Q000235:genetics*; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D010958:Plasminogen / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D013997:Time Factors
D020533:Angiogenesis Inhibitors; D010446:Peptide Fragments; D011994:Recombinant Proteins; D043168:Angiostatins; D010958:Plasminogen