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[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 construct GRα-GST fusion expression vector and obtain recombinant protein, in order to provide a tool for screening glucocorticoid-like Chinese medicine. [Method] The sequence of GRα gene containing CDS was amplified by RT-PCR and inserted into PMD-18 vector by T-A cloning. The recombinant vector was used as a template to amplify GRαORF containing BamH I and Xho I restriction sites. After inserted into pGEX-4T-1 vector, a recombinant vector was constructed and expressed in E. coli BL21 by IPTG induction The fusion protein was detected by SDS-PAGE. [Result] The GRα-complete gene was successfully constructed into prokaryotic expression vector pGEX-4T-1. Sequencing results showed that the vector was successfully constructed without insertions and frameshift mutations. After induction with IPTG, the target protein, GRα-GST fusion protein, which exactly matches the predicted size (112kDa) was obtained. [Conclusion] Escherichia coli with recombinant vector was successfully obtained and expressed under the induction of IPTG. The GRα-GST fusion protein was obtained.