苦荞二氢黄酮醇4-还原酶的原核表达与多克隆抗体制备

Prokaryotic Expression of Tartary Buckwheat Dihydroflavonol 4-reductase and Preparation of Its Polyclonal Antibody

苦荞二氢黄酮醇4-还原酶(DFR)是花青素合成途径的关键酶。该研究以苦荞种子灌浆期cDNA为模板,采用RT-PCR方法克隆苦荞DFR编码基因,并将其连接到表达载体pET47b上,转化获得苦荞DFR编码基因的大肠杆菌BL21(DE3)工程菌,通过IPTG诱导表达,用SDS-PAGE分析表达产物,用亲和层析方法纯化蛋白,制备苦荞DFR多克隆抗体。RT-PCR技术获得了苦荞DFR编码基因的开放阅读框,重组表达载体经PCR和测序鉴定,表明表达载体构建成功,SDS-PAGE分析表达产物分别以可溶和不可溶的形式高效表达,亲和层析纯化得到融合蛋白,Western blotting显示,制备的多克隆抗体能特异识别其对应的抗原,天然的苦荞DFR蛋白在苦荞种子灌浆期中大量表达。苦荞DFR编码基因的原核表达与多克隆抗体的制备,为进一步开展DFR编码基因功能的研究奠定了基础。

Dihydroflavonol 4-reductase（DFR） is a key enzyme in the biosynthesis of anthocyanins in tartary buckwheat. The DFR coding gene was amplified from the seed-filling period eDNA of tartary buckwheat by RT-PCR and ligated to the expression vector pET47b. The E. coli BL21 （DE3） carrying the DFR coding gene was obtained and induced by IPTG. The expression products were analyzed by SDS-PAGE, purified by affinity chromatograph and the high titer polyclonal antiserum raised against rabbit was obtained. The open reading frame of DFR coding gene was obtained by RT-PCR. As analyzed by PCR and DNA sequencing. The recombinant prokaryotic expression vector was constructed successfully. As analyzed by SDS-PAGE, the DFR had been high-efficiency expressed in E. coli in the form of soluble protein and inclusion bodies. Pure fusion protein was obtained by affinity chromatography. Western blotting analysis showed the raised antibody could specifically react with the antigen and native DFR existed in total protein of seed filling period of buckwheat. These results will be valuable for the further study on the biological function of DFR.