[Prokaryotic expression of extracellular ligand binding domains of chick tie-2 and its anti-angiogenesis effect].
OBJECTIVE
To study the prokaryotic expression of extracellular ligand binding domains of chick tie-2, the purification, refolding conditions of the recombinant protein, and its anti-angiogeneic effect.


METHODS
A DNA fragment encoding extracellular ligand binding domains of chick tie-2 was obtained by PCR amplification using a previous constructed plasmid as a template. The amplified fragment was then inserted into prokaryotic expression vector pQE30, and was expressed in E.Coli XL-1 blue by adding isopropyl-beta-D-thiogalactoside(IPTG). The recombinant protein in inclusion bodies was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography under denatured conditions. Then the refolding of the purified protein was performed with gradient dialysis. The target protein was injected s.c. into mouse, and the antibody was detected by ELISA and Western blot analysis. The antibody was purified from the antiserum and then incubated with human umbilical endothelial vein cell (HUEVC) to find its anti-angiogenesis in vitro by using propidium iodide(PI) dying through FACS. Alginate encapsulated tumor cell assays were performed and micro-vessel density was determined by counting per high power field in the sections stained with an antibody reactive to CD31 to test its inhibition of angiogenesis.


RESULTS
The recombinant protein was highly expressed in E.Coli XL-1 blue, and the antibody produced in mouse could specifically recognize the recombinant protein. The purified antibody could induce apoptosis of HUEVC in vitro. The anti-angiogenic effect of the antibody could also be found in alginate-encapsulate tumor cell assay and by counting micro-vessel density.


CONCLUSIONS
The protein of extracellular ligand binding domains of chick tie-2 can be expressed at high level in the prokaryotic expression system, and the expressed protein can induce immune response in mouse. Furthermore, the antibody can induce the anti-angiogenic effect.
Luo
Yan
Y
The Key Laboratory of Biotherapy of Human Diseases, Ministry of Education, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041 PR China.
Wen
Yan-jun
YJ
Tian
Ling
L
Wu
Yang
Y
Liu
Ji-yan
JY
Li
Qiu
Q
Li
Jiong
J
Mao
Yong-qiu
YQ
Deng
Hong-xin
HX
Kang
Bing
B
He
Qiu-ming
QM
Yang
Jin-liang
JL
Wei
Yu-quan
YQ
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002645:Chickens; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000032:analysis; D042787:Receptor, TIE-2 / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology
D020533:Angiogenesis Inhibitors; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011994:Recombinant Proteins; D042787:Receptor, TIE-2