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  

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

作　　者：朱美君[1] 王学臣[1] 陈珈[1] 杜敏 

机构地区：[1]中国农业大学生物学院,北京100094

出　　处：《农业生物技术学报》1999年第3期215-218,共4页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金;国家博士点基金

摘　　要：实验利用ＰＣＲ 技术，从拟南芥质膜水通道蛋白ＲＤ２８ 的ｃＤＮＡ 中扩增了所有植物细胞质膜水通道蛋白的保守区段（４２０ ｂｐ） ，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１ ｍ ｍｏｌ／Ｌ 的ＩＰＴＧ 可诱导表达分子量约４０ ｋＤ 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ＧＳＴＲＤ２８ ，并以纯化的融合蛋白为抗原制备拟南芥质膜水通道蛋白的抗体。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 fusion protein 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.

关 键 词：水通道蛋白 抗体 原核表达 植物细胞质膜 

分 类 号：Q946.1[生物学—植物学] Q942.4