title: [Monoclonal antibody preparation and identification of vascular endothelial growth factor 165 expressed in vitro].
abstract: OBJECTIVE
  To prepare a monoclonal antibody against human vascular endothelial growth factor (VEGF165),  for further study the VEGF165 in the tumorigenesis,  tumor cell migration and the tumor cells escape from the immune response.
  
  
  METHODS
  VEGF165 gene was cloned from the human umbilical vein endothelial cells (HUVEC) by RT-PCR,  and then cloned into the pGEX-6P1,  constructed the prokaryotic expression of pGEX-6P1-VEGF165. The fusion -protein of VEGF165 was expressed in E.coli (BL21) induced by the 1.0 mmol/L IPTG at  37DegreesCelsius after 4 h. The fusion-protein was purified by the MicroSpin GST purification kit for immunized the BALB/c mouse. The monoclonal antibodies (mAbs) against the VEGF165 were prepared by hybridoma technique, and ELISA and Western blot identified their immunoglobulin subclass and specificity. And we used the inhibition the embryo angiogenesis assay,  inhibition the HUVEC migration assay and inhibition the HUVEC tubule information assay to study the bioactivity of the mAbs of VEGF165.
  
  
  RESULTS
  The sequence of the VEGF165 is agreed to the GenBank,  and we obtained five species VEGF165 mAbs,  and the titer of the antibody is high,  and we named,  they are 5A6, 3F5, 6H3, 7D10 and 7A10. Our study showed that the 5A6, 3F5, 6H3, 7D10 were classified to IgG2a,  7A10 was classified to IgG2b,  and the light chain is k.Meanwhile the purified mAbs inhibited formation of chicken embryo blood vessels,  and inhibited tubule formation of the HUVEC and inhibited migration of the HUVEC.
  
  
  CONCLUSIONS
  mAbs against human VEGF165 have the effective bioactivity,  which would play a significant role for further study the mechanism of VEGF165.
authors.affiliation: Department of Traditional Chinese Medicine, Medical School of Yangzhou University, Yangzhou, China. gmkong@yzu.edu.cn
authors.forename: Gui-mei
authors.initials: GM
authors.lastname: Kong
authors.forename: Xiao-rong
authors.initials: XR
authors.lastname: Zhang
authors.forename: Ya-li
authors.initials: YL
authors.lastname: Diao
authors.forename: Ke-yan
authors.initials: KY
authors.lastname: Wu
authors.forename: Qiu-yun
authors.initials: QY
authors.lastname: Liu
authors.forename: Ping
authors.initials: P
authors.lastname: Bo
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D002642:Chick Embryo; D042783:Endothelial Cells / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D018919:Neovascularization, Physiologic / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A