拟南芥质膜水通道蛋白RD28保守区段在大肠杆菌中的表达及其抗体的制备

实验利用ＰＣＲ技术，从拟南芥原膜水旧白ＲＤ２８的ｃＤＮＡ中扩增了所有植物细胞质膜水通道蛋白保守区段（４２０ｂｐ），并将其构入ｐＧＥＸ－ＫＧ。酶切、测序分析表明重组质粒ｐＧＥＸ－ＲＤ２８结构正确。０．１ｍｍｏｌ／Ｌ的ＩＰＴＧ可诱导表达分子量约４０ｋＤ融合蛋白ＧＳＴ－ＲＤ２８，该蛋白主要以非包涵体的形式存在。诱导表达后的菌体超声裂解液经谷胱甘肽活化的Ｓｅｐｈａｒｏｓｅ ４Ｂ亲和层析柱纯化得到了高纯度Ｇ

Expression of Consentative Sections of RD28 in E.coli and Preparation of the Antibody Against RD28

Employing PCR, a 420 bp fragment, which was conserved in all aquapoins of plant plasma membrane, was amplified and cloned into pGEX-KG.Restriction endonuclease analysis and sequence confirmed the corrected construction,and 0.1mmol/L IPTG can induce high expression of GST-RD28 fused protein,which mainly existed in E.coli in soluble form.Employing Glutathione Sepharose 4B column,the expressed GST-RD28 fusionprotein can be absorbed specifically on the column and thus separated from lysates.The absorbed fusion protein was further washed down by glutathione.The purified fusion protein was used to immune rabbits directly and obtained high quality antibody,which provides very useful protein probe for the research about tissue distribution and function analysis of plant aquaporin.