水稻磷酸酯酶2A基因蛋白的原核表达载体构建、表达和纯化

Vector Construction Expression and Purification of Prokaryotic Expression of PP2A Gene of Rice

利用PCR技术,从水稻品种日本晴幼穗cDNA中扩增磷酸酯酶2A的基因ORF片段,并将其克隆入原核表达载体pGEX-6p-1中构建重组质粒pGEX-6p-1-PP-2A,经限制性内切酶酶切鉴定以及测序结果表明成功构建了原核表达载体pGEX-6p-1-PP2A,转化E.coliJM109并通过终浓度为0.4mmol/L的IPTG诱导,表达出39kD的GST-PP2A融合蛋白,并用蛋白亲和层析柱GSTrap FF对表达产物进行纯化,为下一步的研究打下了实验基础。

The rice protein phosphatase- 2A （PP- 2A） gene was amplified by polymerase chain reaction （PCR） from cDNA of young panicle in Nipponbare and cloned into the pGEX - 6P - 1 to construct the recombinant expression vector pGEX - 6p - 1 - PP - 2A . The recombinant was identified by restriction endonuclease digestion and sequencing analysis. E. coli JM109 bearing the plasmid was induced with 0.4mol/L IPTG to express the target protein. The expressed GST - PP2A fusion protein was characterized by SDS - PAGE and purified through the GSTrap FF resin, which would be helpful for further research.