title: [Construction and immunogenicity analysis of tumor peptide vaccine P64k-EGFR(262-328); targeting the dimmerization interface of EGFR].
abstract: OBJECTIVE
  To construct a tumor-specific peptide vaccine P64k-EGFR(262-328); targeting the dimerization interface of EGFR and analyze its immunogenicity in BALB/c mice.
  
  
  METHODS
  The fusion gene of P64k-EGFR(262-328); was amplified by splicing overlap extension-PCR (SOE-PCR) and cloned into the pMD18-T vector. After double-enzyme cleavage and sequence analysis, the fusion gene was cloned into the expression vector pET-21b by digestion with Nde I and Hind III and then transformed into the BL21(DE3). After induced by IPTG to express, the fusion protein P64k-EGFR(262-328); was purified by source Q and Ni-NTA chromatography. BALB/c mice were immunized with the purified protein dissolved in double-distilled water emulsified (1:1) in Freuds' adjuvant, and their sera were tested for antibody titers by ELISA.
  
  
  RESULTS
  The P64k-EGFR(262-328); gene of 2031 bp was acquired. SDS-PAGE showed that the recombinant protein was 70 kDa, expressed mainly in a soluble form. After two steps of column chromatography, the purity was over 95%. The peptide vaccine could elicit a high titer of more than 1:16 000 in BALB/c mice.
  
  
  CONCLUSIONS
  The tumor-specific peptied vaccine P64k-EGFR(262-328); was constructed successfully, which could pay a good foundation for the further study on its function and application.
authors.affiliation: Department of Biotechnology, School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou 510006, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Zhao
authors.forename: Meizhi
authors.initials: M
authors.lastname: Wu
authors.forename: Zhange
authors.initials: Z
authors.lastname: Chen
authors.forename: Huangjin
authors.initials: H
authors.lastname: Li
authors.forename: Lian
authors.initials: L
authors.lastname: Zhou
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000276:immunology*; D019496:Cancer Vaccines / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D010455:Peptides / Q000235:genetics / Q000276:immunology*; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001425:Bacterial Outer Membrane Proteins; D019496:Cancer Vaccines; D010455:Peptides; D011993:Recombinant Fusion Proteins; C112623:lpdA protein, Neisseria meningitidis; D066246:ErbB Receptors