Expression
and Activity Determination of Human Angiostatin (k1-3) in Culture Cells and
Larvae of Silkworm
WANG Ying-Fei*, JIN Yong-Feng,
ZHANG Yao-Zhou
( Institute of Biochemistry, Zhejiang University, Hangzhou 310029,
China )
Abstract 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/2¡Á106
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.
Key words human angiostatin; Bombyx mori
baculovirus expression vector system; endothelisal cells; expression
*Corresponding author: Tel,
86-571-86022648; Fax, 86-571-86971191; e-mail, [email protected]