摘要
利用PCR技术,从甘草质膜水通道蛋白1(plasma membrane intrinsic protein1 from Glycyrrhiza uralensis Fisch,GuPIP1)的cDNA中扩增其保守区段(420bp),并将其构入pGEX-KG。酶切、测序分析表明,重组质粒pGEX-GuPIP1结构正确。IPTG诱导表达分子量约40kDa融合蛋白GST-GuPIP1,该蛋白质主要以非包涵体的形式存在于大肠杆菌中。诱导表达后的菌体超声裂解液经谷胱甘肽亲和层析纯化得到高纯度的GST-GuPIP1,以纯化的融合蛋白为抗原免疫兔子制备甘草质膜水通道蛋白的抗体。抗血清经过纯化后以酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)和Western印迹检测抗体的效价和特异性。结果表明,抗体具有高的效价和特异性。免疫组织细胞化学定位表明,GuPIP1在胚根根尖的表皮细胞和根冠细胞中强烈表达。
Employing PCR, a 420 bp cDNA conserved fragment encoding the plasma membrane intrinsic protein 1 from Glycyrrhiza uralensis Fisch （GuPIP1） was PCR amplified and subcloned into pGEX-KG. Restriction endonuclease analysis and sequence confirmed the corrected construction, and IPTG can induce high expression of 40 kDa GST-GuPIP1 fused protein, which mainly existed in E. coli in soluble form. Employing Glutathione Sepharose 4B column, the expressed GST-GuPIP1 fusion protein can be absorbed specifically on the column and thus separated from lysates. The purified fusion protein was used to immune rabbits directly. The antisera were purified and characterized by ELISA and Western blot. The results showed that the antibodies had high titer and specificity. GuPIP1 was abundantly expressed in epidermis cells and root cap cells of Glycyrrhiza uralensis Fisch radicles through immunocytochemical method.
出处
《细胞生物学杂志》
CAS
CSCD
2009年第5期665-670,共6页
Chinese Journal of Cell Biology
基金
福建省自然科学基金资助项目(No.2008J04011)
福建省高校服务海西建设重点项目(No.A101)
福建省教育厅科技计划项目(No.2007JA07151)
泉州市科技局技术研究与开发项目(No.2007N6)
泉州师范学院重点学科建设经费资助
泉州师范学院大学生基金资助项目(No.2008DA008和No.2008DA009)~~
