Expression of human endostatin in larvae of silkworm (Bombyx mori) and in vitro activity assays.
Human endostatin is a novel antiangiogenic molecule, which can inhibit the proliferation and development of new blood vessels, and experimentally can cause nearly complete regression of established tumors. In this paper, the cDNA encoding human endostatin was cloned into a baculovirus shuttle vector pBacPAK8 and co-infected with linearized Bm-BacPAK6 DNA into and BmN cells. The recombinant virus was screened and identified by PCR, DNA and RNA dot hybridization, and ELISA assay. The recombinant endostatin was expressed in culture cells, and the larvae and pupa of silkworm by inoculation of recombinant virus. The biological activity assay showed that the expression product in larvae was over 150 microg/ml, about 50-fold higher than that expressed in cultured cells. SDS-PAGE and Western blotting analysis showed a pattern of molecular weight of about 20 kDa. The bio-activity of the protein product was determined by human umbilical vein endothelial cells (ECV304) proliferation test in vitro and the chick chorioallantoic membrane (CAM) vascular inhibition test. Endostatin showed significant inhibitory effect on endothelial cells in a dose-dependent manner. Silkworm-produced endostatin induced apoptosis of endothelial cells and also inhibited angiogenesis in the CAM assay. Combination regimen using angiostatin and endostatin showed more than additive effect in angiogenic inhibition and increasing apoptosis when compared with treatment with the individual antiangiogenic protein.
Institute of Biochemistry, Zhejiang University, Hangzhou, People's Republic of China.
Jin
J
Yongfeng
Wang
W
Yingfei
Zhu
Z
Zhenhong
Zhang
Z
Yaozhou
D043168:Angiostatins; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D012831:Bombyx; D002455:Cell Division; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis* / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D043169:Endostatins; D004730:Endothelium, Vascular / Q000166:cytology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008854:Microscopy, Electron; D009389:Neovascularization, Pathologic; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000494:pharmacology; D010958:Plasminogen / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology
D018076:DNA, Complementary; D043169:Endostatins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D043168:Angiostatins; D010958:Plasminogen; D003094:Collagen