摘要
[目的]构建GRα-GST融合表达载体并获得重组蛋白,以期为筛选类糖皮质激素样中药提供工具。[方法]运用RT-PCR扩增得到包含CDS的GRα基因序列后,经T-A克隆插入PMD-18载体。以此重组载体为模板扩增含有BamH I与Xho I酶切位点的GRαORF,插入pGEX-4T-1载体后形成了重组载体,并使其在大肠杆菌BL21中经IPTG诱导表达融合蛋白,再用SDS-PAGE检测融合蛋白的表达。[结果]成功的将GRα-全基因构建入原核表达载体pGEX-4T-1中。测序结果表明载体构建成功,无插入和移码突变。经IPTG诱导后获得与预测大小(112kDa)完全一致的目的蛋白即GRα-GST融合蛋白。[结论]成功获得了含重组载体的大肠杆菌,并在IPTG诱导下较好的表达,得到了GRα-GST融合蛋白。
[Objective] To structure fusion protein GRα-GST for screening the glucocorticoid-like Chinese medicines.[Methods] Amplified the GR α gene sequence containing CDS by RT-PCR and then ligated to T-A vector PMD-18.Amplified the GR α ORF containing BamH I and Xho I site on the template of the recombinant PMD-18.Fusion protein GRα-GST was expressed in E.coli（BL21） and induced by IPTG.[Results] GRα sequence was inserted to pGEX-4T-1 vector and the fusion protein GRα-GST was expressed successfully.[Conclusion] We obtained the recombinant plasmid（pGEX-4T-1GRα）,and fusion protein GRα-GST could be expressed in E.coli by the induction of IPTG.
出处
《浙江中医药大学学报》
CAS
2012年第11期1211-1213,1217,共4页
Journal of Zhejiang Chinese Medical University
基金
浙江省教育厅科研项目资助(Y200908682)
浙江省中医药科学研究基金计划(2010ZB028)~~
作者简介
通讯作者：谢志军，E—mail：xzj575@163．com
