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目的:优化β-淀粉样蛋白单链抗体(scFv)的诱导表达和纯化条件。方法:在固定诱导表达时间和诱导表达温度的条件下,采用不同终浓度的IPTG进行诱导表达,通过SDS-PAGE分析全菌液总蛋白中目的蛋白E3 scFv的表达水平,确定所需诱导剂IPTG的最佳浓度。在目的蛋白结合到亲和层析柱之后,采用不同终浓度的咪唑溶液进行洗脱,通过收集各管洗脱液、Western blot等方法确定预洗脱液咪唑的最佳浓度。结果:E3 scFv在20℃诱导表达18 h时,所需诱导剂IPTG的最佳浓度为0.1 mmol/L;纯化时,预洗脱液咪唑的最佳浓度为10 mmol/L。结论:通过优化上述条件,我们建立了一个高效表达及纯化E3 scFv的方法,为后续的研究奠定了基础。
Objective: To optimize the induction and purification of β-amyloid single chain antibody (scFv). Methods: IPTG with different final concentrations was induced under fixed induction time and induction temperature, and the expression level of target protein E3 scFv in total protein of whole bacteria was analyzed by SDS-PAGE. The required inducer IPTG The best concentration. After the target protein was bound to the affinity column, the imidazole solution with different final concentrations was used for elution. The optimal concentration of imidazole in the pre-eluted solution was determined by collecting the eluent of each tube and Western blot. Results: The optimum concentration of IPTG for inducing E3 scFv was induced at 20 ℃ for 18 h, and the optimum concentration of imidazole was 10 mmol / L. Conclusion: By optimizing the above conditions, we established a method for efficient expression and purification of E3 scFv, which laid the foundation for further research.