Prokaryotic expression and purification of antifreeze protein DAFP and preparation of its antiserum

AIM: 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 mass (M_r) 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. CONCLUSION: The DAFP was expressed in E.coli and its specific antibody was prepared successfully.