Molecular cloning of dihydroflavonol 4-reductase gene from grape berry and preparation of an anti-DFR polyclonal antibody

文献类型： 外文期刊

第一作者： Zhang, Ping

作者： Zhang, Ping;Wen, Peng-Fei;Wan, Si-Bao;Wang, Wei;Pan, Qiu-Hong;Zhan, Ji-Chen;Huang, Wei-Dong;Zhang, Ping;Wen, Peng-Fei

作者机构：

关键词： DFR;Escherichia coli expression;antibody..;purification;grape berry

期刊名称：VITIS （ 影响因子：1.339； 五年影响因子：1.306 ）

ISSN： 0042-7500

年卷期： 2008 年 47 卷 3 期

页码：

收录情况： SCI

摘要： Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) is a key enzyme of the flavonoid pathway, which synthesizes numerous secondary metabolites to determine the quality of grape berry and wine. The full-length dfr cDNA wit h 1014 bp was cloned from grape berry, and then introduced into an expressed plasmid pET-30a (+) vector at the EcoR I and Xho I restriction sites. With induction of the isopropyl-beta-D-thiogalactoside (IPTG), the pET-dfr was highly expressed in Escherichia coli BL21 (DE3) pLysS cells. A fusion protein with the His-Tag was purified through Ni-NTA His Bind Resin and then used as the antigen to immunize a New Zealand rabbit. The resulting antiserum was further purified precipitated by 50 % saturated ammonium sulfate and DEAE-Sepharose FF chromatography to obtain the immunoglobulin G (IgG) fraction. The resulting polyclonal antibody was found capable of immuno-recognizing the DFR of the crude protein extracts from grape berry. This work undoubtedly provides the possibility for further studies on biological regulation of DFR activity in grape berry.

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