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作 者 ：李玉萍,邓丹丹,张海纳,李学俊*,陈 鹏

期 刊 ：西北植物学报 2013年 4期 页码：672~677

关键词：苦荞；黄酮-3-羟化酶；原核表达；多克隆抗体；

Keywords:tartary buckwheat, flavanone-3-hydroxylase, prokaryotic expression, polyclonal antibody,

摘 要 ：为了制备苦荞黄酮-3-羟化酶的多克隆抗体,该研究以苦荞种子灌浆期cDNA文库中获得的苦荞黄酮-3-羟化酶基因截短体（truncated Flavanone-3-hydroxylase,TrF3H）序列为基础,采用PCR扩增F3H的截短序列编码区（TrF3H）,构建了原核表达载体pET47b-TrF3H,并转化入大肠杆菌Rosetta(DE3)plysS中进行诱导表达,将经钴离子螯合层析柱纯化后的目的蛋白切胶回收后制备了高效价的多克隆抗体。结果表明：pET47b-TrF3H在大肠杆菌Rosetta(DE3)plysS中以包涵体的形式高效表达。蛋白质印迹显示，制备的多克隆抗体能特异识别其对应的抗原,天然的黄酮-3-羟化酶蛋白在苦荞的未成熟种子中大量表达。原核表达体系的建立和多克隆抗体的制备为进一步探讨F3H在苦荞中功能奠定了基础。

Abstract:In the base of truncated Flavanone-3-hydroxylase gene (TrF3H) achieved from full-length cDNA library of tartary buckwheat which was in seed-filling period,prokaryotic expression vector pET47b-TrF3H with truncated coding region of F3H（TrF3H）was constructed and transformed into E.coli Rosetta (DE3) plysS.After the overexpressed target protein purified by cobalt chelating chromatography,the high titer polyclonal antiserum raised against rabbit was obtained.The results demonstrated that the TrF3H had been expressed in E.coli in the form of inclusion bodies and western blotting analysis showed the raised antibody could specifically react with the antigen while native F3H existed in immature seed protein.These results laid the basis for further exploration of the functions of F3H in tartary buckwheat.

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