| OsMAPK4基因的原核表达及蛋白纯化 |
| |
| 引用本文： | 刘西燕,柏锡,李莹,李杰.OsMAPK4基因的原核表达及蛋白纯化[J].东北农业大学学报,2008,39(8). |
| |
| 作者姓名： | 刘西燕 柏锡 李莹 李杰 |
| |
| 作者单位： | 东北农业大学生命科学学院,哈尔滨,150030 |
| |
| 基金项目： | 国家自然科学基金，黑龙江省教育厅科学技术研究项目 |
| |
| 摘 要： | 构建OsMAPK4基因的原核表达载体,对表达产物进行鉴定和纯化,为转OsMAPK4基因植株后期的Western Blot检测提供抗原。用PCR方法从本实验室已构建的植物表达载体pUM4上扩增OsMAPK4基因,将其克隆至pET32b原核表达载体,构建重组载体pET32b-MAPK4。转化大肠杆菌BL21,IPTG诱导表达后进行SDS-PAGE分析。用Ni-NTA亲和层析柱对重组蛋白进行纯化,用Western Blot进行蛋白鉴定。结果表明,原核表达载体构建正确;SDS-PAGE分析及Western Blot鉴定中,均出现了与DNAMAN预测的43.6 ku大小一致的蛋白条带;得到纯化蛋白。成功构建了OsMAPK4基因的原核表达载体,得到纯化蛋白,为转OsMAPK4基因水稻植株体内蛋白表达活性的检测提供了抗原。
|
| 关 键 词： | OsMAPK4基因 原核表达 纯化 Western Blot |
Prokaryotic expression of OsMAPK4 gene and purification of the protein |
| |
| LIU Xiyan,BAI Xi,LI Ying,LI Jie.Prokaryotic expression of OsMAPK4 gene and purification of the protein[J].Journal of Northeast Agricultural University,2008,39(8). |
| |
| Authors: | LIU Xiyan BAI Xi LI Ying LI Jie |
| |
| Abstract: | This paper is aimed to construct a prokaryotic expression vector for OsMAPK4 gene,identify the expressed product,and obtain the purification protein.Amplified OsMAPK4 gene by PCR from pUM4 vector which had been constructed in our laboratory.Clone amplified OsMAPK4 gene into pET32b prokaryotic expression vector.The new vector was named pET32b-MAPK4.Transformed the constructed recombinant plasmid to E.coli for expression under induction of IPTG.Identified the expressed product by SDS-PAGE and Western Blot.Purified the protein with Ni-NTA Purification System.Both PCR and DNA sequencing proved that recombinant plasmid was correctly constructed.Both SDS-PAGE and Western Blot showed positive bands consistented with those expected.The prokaryotic expression vector for OsMAPK4 gene was successfully constructed,the purification was obtained,which provided the antigen for Western Blot of the transgenic rice plant. |
| |
| Keywords: | Westem Blot |
| 本文献已被 维普 万方数据 等数据库收录！ |
|
