液泡膜水通道蛋白γ-TIP表达载体的构建及其抗体的制备  

The Construction of γ-TIP Prokaryotic Expression Vector and Preparation of the γ-TIP Antibody

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：《Acta Botanica Sinica》1999年第11期1235-1238,共4页Acta Botanica Sinica（植物学报：英文版）

基　　金：国家自然科学基金!39600090;39730050;国家重点基础研究发展规划资助

摘　　要：利用PCR技术，从拟南芥（Arabidopsisthaliana（L．）Heynh．）液泡膜水通道蛋白γ－TIP的cDNA中扩增了含水通道特异保守区的205bp片段，并将其构入pGEX－KG原核表达载体。酶切、测序分析表明构建的重组质粒pGEX-TIP结构正确。0．4mmol/L的IPTG可诱导表达分子量为32kD融合蛋白GST-TIP，表达蛋白以包涵体形式存在，约占菌体总蛋白的50％。实验用SDS-PAGE制备电泳从菌体的超声裂解液中纯化了GST-TIP融合蛋白，以此融合蛋白为抗原制备了高质量的液泡膜水通道蛋白的抗体，为研究水通道蛋白在组织中的定位及生理功能提供了有用的蛋白探针。A prokaryotic expression vector, pGEX-TIP, was constructed from Arabidopsis thaliana (L. )Heynh. Employing PCR, 205 bp fragment near 3' end of γ-TIP cDNA, which has specific aquaporin activity,was amplified and cloned into pGEX-KG. Restriction endonuclease analysis and sequencing confirmed the cormct construction, and 0.4 mmol/L IPTG can induce high expression of GST-TIP fused protein which was about 50% in total of E. coli proteins. The IPTG induced E. coli was collected and lysed by supersonic heatment. The fusion protein was mainly recovered as an inclusion body. The expressed GST-TIP was purified by SDS-PAGE according to their molecular weight, which was about 32 kD. The purified protein was used to immune dsits dimetly or was electworetically eluted before it was used for inunnization. The highly qualilied antimp for GST-TIP was obtained, which provides a very useful protein probe for the research on localization and function of aquaporins.

关 键 词：水通道蛋白 抗体 原核表达 载体构建 液泡膜 

分 类 号：Q78[生物学—分子生物学] Q942.6