【摘要】 目的在原核中高效表达及活性鉴定重组人survivin蛋白分子。方法应用RT-PCR的方法得到survivin的cDNA,并构建成pBV220-survivin原核表达质粒,将其导入E.coliBl21(Gold)中,诱导表达survivin蛋白。通过DEAESepharoseFastFlow离子交换柱和SephacrylS-200分子筛二步柱纯化以获取目的蛋白。用Westernblotting检测表达产物。结果PT-PCR产物经测序分析与预期结果完全一致,表达质粒在E.coliBl21(Gold)中得到高效表达,表达的survivin蛋白占总蛋白含量的30%以上,表达产物以包涵体的形式存在。通过离子交换柱和分子筛二步柱纯化及复性后获得的目的蛋白,其纯度达到95%以上。Westernblotting证明表达产物具有特异性。结论获得了较高纯度的survivin蛋白,为进一步深入研究该蛋白的细胞凋亡调控功能提供了工具。更多还原

【Abstract】 Objective To efficiently express and identify recombinant human survivin in E. coli. Methods Survivin cDNA was amplified by reverse transcriptional (RT)-PCR and cloned into the prokaryotic expression vector pBV220, followed by expression of the recombinant plasmid in E.coli strain BL21 (Gold). To obtain survivin protein, DEAE-Sepharose Fast-Flow ion exchange chromatography and Sephacryl S-200 gel filtration were performed. Western blot analysis was used for detecting the expressed product. Results Survivin protein was expressed in E.coli in the form of inclusion body at the expression level over 30% of the total cell protein. After ion exchange chromatography and gel filtration, the recombinant protein reached a purity over 95% and exhibited specific reaction with mouse anti-human antibody. Conclusion Survivin protein with high purity can be obtained by the method described above to facilitate further study of the anti-apoptosis function of survivin.更多还原