CTB-Aβ42融合蛋白的原核表达条件优化及鉴定

摘要

目的 探索融合基因CTB-Aβ42在大肠杆菌中表达的最适条件,并纯化得融合蛋白.方法 设置不同的诱导温度、诱导时间、异丙基-β-D-硫代半乳糖苷(IPTG)浓度,在大肠杆菌中表达融合基因CTB-Aβ42,SDS-PAGE凝胶电泳鉴定表达产物,GST亲和色谱柱纯化目的蛋白,Western blot鉴定目的蛋白免疫原性.结果 SDS-PAGE结果显示,GST-CTB-Aβ42融合蛋白分子质量约为45 kD,与预期结果一致;Western blot结果表明,纯化得到的目的蛋白具有免疫原性.在诱导条件为30℃,0.1 mmol/L IPTG诱导2h表达的可溶性蛋白所占比例最高;在37℃,0.1mmol/L IPTG诱导4h表达的融合蛋白总量最高,小部分可溶性表达,大部分以包涵体的形式存在.结论 本实验对CTB-Aβ42融合蛋白的原核表达条件进行了优化,经GST亲和色谱柱纯化获得具有免疫原性的融合蛋白.%Purpose To explore the optimal conditions of the expression of fusion gene CTB-AS.42 in E. Coli,and to purify the fusion protein CTB-Ap42. Methods Induced expression of fusion protein CTB-Ap42 in E. Coli of different temperatures, time and concentration of 1PTG. The expression product was determined by SDS-PAGE and Western blot analysis. Fusion protein was purified by GST affinity chromatog-raphy. Results SDS-PAGE results showed that the molecular mass of fusion protein GST-CTB-Ap42 is 45 kD as expected, and Western blot analysis indicated that anti- CTB antibody could react specifically against the fusion protein. Soluble expression reaches the highest level when it is induced by 0. 1 mmol/L IPTG as final concentration for 2 h at 30℃. If induced by 0.1 mmol/L IPTG as final concentration for 4 h at 37℃, intracellular expression as inclusion bodies was the most efficient. Conclusion We optimized the conditions of the expression of fusion protein CTB-Aβ42 in E. Coli,and acquired purified fusion protein relatively. It provides a sufficient foundation for the further study on vaccine against Alzheimer disease.