title: [Prokaryotic expression of extracellular ligand binding domains of chick tie-2 and its anti-angiogenesis effect].
abstract: 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.
authors.affiliation: The Key Laboratory of Biotherapy of Human Diseases, Ministry of Education, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041 PR China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Luo
authors.forename: Yan-jun
authors.initials: YJ
authors.lastname: Wen
authors.forename: Ling
authors.initials: L
authors.lastname: Tian
authors.forename: Yang
authors.initials: Y
authors.lastname: Wu
authors.forename: Ji-yan
authors.initials: JY
authors.lastname: Liu
authors.forename: Qiu
authors.initials: Q
authors.lastname: Li
authors.forename: Jiong
authors.initials: J
authors.lastname: Li
authors.forename: Yong-qiu
authors.initials: YQ
authors.lastname: Mao
authors.forename: Hong-xin
authors.initials: HX
authors.lastname: Deng
authors.forename: Bing
authors.initials: B
authors.lastname: Kang
authors.forename: Qiu-ming
authors.initials: QM
authors.lastname: He
authors.forename: Jin-liang
authors.initials: JL
authors.lastname: Yang
authors.forename: Yu-quan
authors.initials: YQ
authors.lastname: Wei
mesh_terms: 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
chemical_list: D020533:Angiogenesis Inhibitors; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011994:Recombinant Proteins; D042787:Receptor, TIE-2