[Expression and biological activity of human angiogenesis inhibitor angiostatin in insect cells].
OBJECTIVE
Angiogenesis is the essential process for tumor growth and metastasis. Angiostatin, a potent endogenous inhibitor of angiogenesis, could selectively inhibit proliferation of vascular endothelial cells. This study was to construct recombinant angiostatin-baculovirus, and explore the expression and biological activity of recombinant angiostatin in insect cells.


METHODS
The angiostatin baculovirus transfer vector pBlueBacHis2B was co-transfected with virus DNA into insect Sf9 cells to construct recombinant baculovirus. The recombinant virus was screened by plaque assay, and confirmed and amplified by polymerase chain reaction (PCR) to produce large scale, high-titer virus stock. The expression of the recombinant protein at different time points was detected by SDS-PAGE and Western blot. The recombinant protein was purified by ProBond purification system. Inhibitory effect of recombinant angiostatin on human umbilical vein endothelial cells (HUVECs) was examined by MTT assay. Its anti-angiogenesis effect was confirmed by in vivo chorioallantoic membrane (CAM) test.


RESULTS
Recombinant angiostatin baculovirus with a high virus titer (2 x 10(8) pfu/ml) was constructed successfully. Recombinant angiostatin (53 ku) was effectively expressed in Sf9 cells, and its pure degree was about 90% of insect cellular total soluble proteins. The recombinant angiostatin obviously inhibited proliferation of endothelial cells with the 50% inhibitory concentration (IC(50)) of 2.3 microg/ml, and remarkably inhibited the growth of vessels in CAM.


CONCLUSIONS
The baculovirus expression system could be used to construct high-titer recombinant angiostatin-virus stock, and highly express the recombinant angiostatin in insect cells. In vitro and in vivo experiments confirmed that angiostatin could inhibit proliferation of vascular endothelial cells.
Xu
Xiao-Lin
XL
Liaoyang School of Chinese Traditional Medicine, Liaoyang, Liaoning, P.R. China.
Liu
Ge-Fei
GF
Huang
Dong-Yang
DY
D020533:Angiogenesis Inhibitors / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D043168:Angiostatins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000098:blood supply / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology; D005822:Genetic Vectors; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism*; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology
D020533:Angiogenesis Inhibitors; D011994:Recombinant Proteins; D043168:Angiostatins