水稻生长素结合蛋白ABP1的原核表达及纯化

摘要

The auxin binding protein could be used as a highly specific and affinitive recognizing molecule in the immunoassay and biosensors to detect auxin because of its specific binding capability to the auxin. Rice( Oryza sativa) auxin binding protein 1 gene obtained by RT-PCR was cloned into the prokaryotic expression vector pET-32a( + ) to construct the recombinant plasmid pET-32a-ABP1. After digestion, PCR and DNA sequencing identification, the recombinant plasmid was transformed into expressional bacteria E. coil BL21 ( DE3 ), then the ABP1 protein induced by IPTG and purified by affinitive chromatography was expressed successfully and identified by SDS-PAGE and Western Blot. Conclusion: We have successed in expressing a soluble fusion protein about 40 kDa irithe E. coil BL21 ( DE3 ) and obtained the purified ABP1 protein with Ni2 + -NTA affinitive column.%生长素结合蛋白能够与生长素特异性结合,因而有可能直接被用作生长素免疫分析和生物传感测定中的高特异性、高亲和力识别分子.本研究通过RT-PCR获得水稻生长素结合蛋白1(ABP1)cDNA,将其克隆到原核表达载体pET-32a(+)中,成功构建pET-32a-ABP1 重组表达载体.经酶切、PCR及DNA测序鉴定后,将阳性质粒转化表达受体菌E.coil B121(DE3).加入异丙基-β-D-硫代半乳糖苷(1PTG)进行诱导后,取样进行SDSPAGE和Western Blot分析.结果表明成功表达出一个分子量约为40 kD的可溶性融合蛋白,并利用Ni2+-NTA亲和柱一步纯化方式得到了ABP1.