title: Cloning of variable fragments of tumor immunoglobulin, assembling and expressing of human SCFV protein in E. coli for anti-idiotype vaccination.
abstract: OBJECTIVE
  Idiotype, the unique part of immunoglobulin molecule expressed on the surface of B-cells, represents a specific antigen for vaccination against lymphoma. We have developed a rapid method for immunoglobulin variable fragments cloning, assembling and expression of recombinant idiotype protein in Escherichia coli.
  
  
  METHODS
  PCR with specially designed panel of primers was used for direct amplification of variable regions of tumor immunoglobulin. Overlapping extension PCR, restriction and ligation was applied for assembling and cloning of vaccine construction. Idiotype protein was purified by metal-chelate chromatography.
  
  
  RESULTS
  Methods of idiotype cloning from lymphoma cells and production of recombinant protein were developed and optimized. Several samples of idiotypic proteins originating from B-cell lines and lymphoma patients were produced.
  
  
  CONCLUSIONS
  The proposed method of vaccine production is relatively cheap, not very laborious and requires as long as 6-7 week to perform. The expressed protein was soluble, did not accumulate in inclusion bodies and harvested at sufficient for vaccination quantity and concentration.
authors.affiliation: Belarusian Research Center for Pediatric oncology & Hematology, Minsk, Belarus. meleshko@tut.by
authors.forename: A N
authors.initials: AN
authors.lastname: Meleshko
authors.forename: K P
authors.initials: KP
authors.lastname: Vashkevich
authors.forename: E G
authors.initials: EG
authors.lastname: Fomina
authors.forename: E P
authors.initials: EP
authors.lastname: Scheslenok
authors.forename: T V
authors.initials: TV
authors.lastname: Schkolina
authors.forename: G V
authors.initials: GV
authors.lastname: Sergeev
mesh_terms: D000912:Antibodies, Neoplasm / Q000235:genetics* / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D019496:Cancer Vaccines / Q000235:genetics / Q000276:immunology; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005784:Gene Amplification; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology; D007130:Immunoglobulin Idiotypes / Q000235:genetics* / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D007075:Immunoglobulin M / Q000235:genetics / Q000276:immunology; D007145:Immunoglobulin kappa-Chains / Q000235:genetics / Q000276:immunology; D016393:Lymphoma, B-Cell / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology
chemical_list: D000912:Antibodies, Neoplasm; D019496:Cancer Vaccines; D007074:Immunoglobulin G; D007130:Immunoglobulin Idiotypes; D007147:Immunoglobulin Light Chains; D007075:Immunoglobulin M; D007145:Immunoglobulin kappa-Chains; D057127:Single-Chain Antibodies