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2 h表达的可溶性蛋白所占比例最高;在37℃,0.1mmol/L IPTG诱导4 h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-Aβ42 in E.Coli,and to purify the fusion protein CTB-Aβ42.Methods Induced expression of fusion protein CTB-Aβ42 in E.Coli of different temperatures,time and concentration of IPTG.The expression product was determined by SDS-PAGE and Western blot analysis.Fusion protein was purified by GST affinity chromatography.Results SDS-PAGE results showed that the molecular mass of fusion protein GST-CTB-Aβ42 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.
出处
《中国生化药物杂志》
CAS
CSCD
北大核心
2012年第4期342-345,349,共5页
Chinese Journal of Biochemical Pharmaceutics
基金
浙江省自然科学基金面上项目(Y3110354和Y3090339)
浙江省教育厅科研项目(Y200909740)
生物科学实验教学示范中心项目(SB1108001-E)
