Wang Zhuanhua, Wang Lan, Chang Wenjun, Li Yuying, Zhang Zheng, Wieslander Gunilla, Norback Dan
Institute of Biotechnology, Shanxi University, Taiyuan, China.
Biosci Biotechnol Biochem. 2006 May;70(5):1195-9. doi: 10.1271/bbb.70.1195.
A cDNA fragment encoding a 24 kDa allergenic protein in tartary buckwheat was obtained using reverse transcription PCR, 3'-rapid amplification of cDNA ends (RACE), and nest PCR. The cDNA clone contained 768 nucleotides, including 588 nucleotides in the open reading frame (ORF) and 180 nucleotides in the 3'-terminal sequence. The ORF encoded a functional protein of 195 amino acids. It shared 95% and 93% nucleotide homology with the allergenic storage protein and a legumin-like protein from common buckwheat respectively. The encoding region was expressed in host strain Escherichia coli BL21 (DE3) induced by IPTG at 28 degrees C. The inclusion bodies of recombinant protein obtained were analyzed by western blot and purified by affinity chromatography. The purity of target protein reached above 95%. After they were refolded by step-wise dialysis, 68% of the inclusion bodies reached soluble state. An analysis of immunological activity showed that the recombinant protein had a specific IgE binding activity. This is the first report of the molecular cloning and expression of the major allergen from tartary buckwheat.
利用逆转录聚合酶链反应（RT-PCR）、cDNA末端快速扩增技术（3'-RACE）和巢式PCR，获得了一个编码苦荞中24 kDa变应原蛋白的cDNA片段。该cDNA克隆包含768个核苷酸，其中开放阅读框（ORF）有588个核苷酸，3'-末端序列有180个核苷酸。该ORF编码一个由195个氨基酸组成的功能蛋白。它与苦荞的变应原储存蛋白和类豆球蛋白分别有95%和93%的核苷酸同源性。编码区在28℃下经异丙基-β-D-硫代半乳糖苷（IPTG）诱导，在宿主菌株大肠杆菌BL21（DE3）中表达。对获得的重组蛋白包涵体进行蛋白质免疫印迹分析，并通过亲和层析进行纯化。目标蛋白的纯度达到95%以上。经逐步透析复性后，68%的包涵体达到可溶状态。免疫活性分析表明，该重组蛋白具有特异性IgE结合活性。这是关于苦荞主要变应原分子克隆与表达的首次报道。
