[Prokaryotic expression and purification of antifreeze protein DAFP and preparation of its antiserum].
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.
Wang
Yun
Y
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.
Lu
Guo-dong
GD
Xue
Na
N
Zhang
Fu-chun
FC
Ma
Ji
J
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
D021301:Antifreeze Proteins; D007106:Immune Sera; D019476:Insect Proteins