title: [Prokaryotic expression and purification of antifreeze protein DAFP and preparation of its antiserum].
abstract: OBJECTIVE
  To prepare the Dendroides canadensis antifreeze proteins (DAFP) specific mouse antiserum.
  
  
  METHODS
  According to the published antifreeze protein gene (gi: 2737939) from Dendroides canadensis in GenBank, one isoform of the gene was chemically synthesized and cloned into the expression vector pGEX-4T-1 and pMAL-p2x respectively and expressed in E.coli. The expressed fusion protein was purified through Glutathione Sepharose 4B column, and then used to immunize the mice for preparing the specific the antibody. The titer and specificity of the antibody were analyzed by ELISA and Western blot, respectively.
  
  
  RESULTS
  SDS-PAGE analysis showed that the DAFP was expressed in E.coli. The relative molecular masses of expressed fusion protein was 38,000. The titer of antibody was 1:5,000. Western blot analysis showed the expressed protein of BL21/pGEX-4T-1-dafp and TB1/pMAL-p2x-dafp could react specifically with DAFP antibody.
  
  
  CONCLUSIONS
  The DAFP was expressed in E.coli and its specific antibody was prepared successfully.
authors.affiliation: Key Laboratory of Molecular Biology, College of Life Science and Technology, Xinjiang University, Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, Urumqi 830046, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Wang
authors.forename: Guo-dong
authors.initials: GD
authors.lastname: Lu
authors.forename: Na
authors.initials: N
authors.lastname: Xue
authors.forename: Fu-chun
authors.initials: FC
authors.lastname: Zhang
authors.forename: Ji
authors.initials: J
authors.lastname: Ma
mesh_terms: D000818:Animals; D021301:Antifreeze Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D001517:Coleoptera / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007106:Immune Sera / Q000276:immunology*; D019476:Insect Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D051379:Mice
chemical_list: D021301:Antifreeze Proteins; D007106:Immune Sera; D019476:Insect Proteins