摘要
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。
AIM： To optimize the expression and purification protocol for human scFv antibody againstmyloid peptide. METHODS： Expression of E3 scFv was induced by different concentrations of IPTG under the fixed condition of time period and temperature, and the optimal concentration of IPTG was determined by SDS-PAGE analysis on E3 scFv expression level. Furthermore, elution buffer with different concentrations of imidazole was used for pre-eluting to determine the optimal pre-eluting condition by Western blotting against E3 scFv. RESULTS： We obtained the highest expression of E3 scFv after 18 h induction with 0.1 mmoVL IPTG under 20℃. In addition, Western blotting indicated the highest purity of E3 scFv when the resin was pre-eluted with the buffer containing 10 mmol/L imidazole. CONCLUSION： Through optimizing the above mentioned conditions, we established an improved strategy for high expression and efficient purification of E3 scFv, which paves the way for further research.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2012年第7期718-721,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81172119)
关键词
Β-淀粉样蛋白
单链抗体
表达纯化
条件优化
β-amyloid peptide
single-chain antibody
expression and purification
condition optimization
