[Cloning, expression of soluble VEGFR2 fragment and its effect on tumor angiogenesis].
OBJECTIVE
To study the anti-tumor angiogenesis effect of soluble VEGF receptor fragment by blocking the combination of VEGF and its receptor in vivo and in vitro.


METHODS
RT-PCR technique was used to amplify Flk-1/KDR fragment from embryo mouse liver, which was recombinated to expression vector pET-28b(+) and retrovirus vector PLXSN, which was induced to be expressed, purified and identified with EcoR I and Hind III. Mouse endothelial cells were separated, cultured and identified by immunocytochemistrical staining using VIII factor-related antigen antibody. The expressed product was analyzed about its effect on endothelial cell's growth in vitro with MTT method. The retrovirus vector was transfected to tumor cell lines S180 and B16 by liposome method to observe the biological specificity in vitro after gene transfection.


RESULTS
1000 bp size sVEGFR fragment was amplify from E9, E11 embryo mouse liver tissues, which was recombinated to TA clone vector and identified by sequence analysis. This fragment was cloned to expression vector pET-28b(+), the expressed product was purified and identified correctly. The in vitro study showed this expressed product can effectively inhibit endothelial cell(s), growth and proliferation. The fragment was then cloned to retrovirus vector PLXSN and transfected to tumor cell lines S180 and B16 successfully with RT-PCR and SDS-PAGE. The experiments in vivo showed that the weight of tumor smaller, the size decreased significantly, the microvessel density was fewer and Flk1 protein expression were higher in the group of gene transfection than that of control.


CONCLUSIONS
Soluble VEGFR fragment is a kind of effective gene engineer product for anti-tumor angiogenesis gene therapy and the development of anti-tumor drug.
Kou
Bai-jun
BJ
Laboratory of Pathobiology, College of Basic Medicine, Jilin University 130021, China. koubaijun@yahoo.com
Li
Yu-lin
YL
Zhang
Li-hong
LH
Zhu
Gui-bin
GB
Wang
Xin-rui
XR
Li
Yi-lei
YL
Wang
Yue-zeng
YZ
Shi
Ying-ai
YA
D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular; D042783:Endothelial Cells / Q000166:cytology; D005822:Genetic Vectors; D008546:Melanoma, Experimental / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic / Q000473:pathology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D012510:Sarcoma 180 / Q000378:metabolism / Q000473:pathology*; D014162:Transfection; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology
D012333:RNA, Messenger; D040301:Vascular Endothelial Growth Factor Receptor-2