重组胸腺素α_1的分离纯化和活性测定

Purification and biological activity of recombinant thymosin alpha1

目的 利用融合表达载体 p Thio His A的重组质粒p Thio His A- Tα1 4,转化大肠杆菌 TOP10得到的工程菌 ,来分离纯化表达的胸腺素 α1 (thym osin alpha1,Tα1 ) .方法 将高效表达融合蛋白的工程菌用细胞破碎器裂解 ,经 80℃热处理 ,离心上清采用 Q- Sepharose FF阴离子交换色谱纯化融合蛋白 .融合蛋白经 CNBr裂解后用常压离子交换色谱纯化Tα1 单体 .应用 SDS- PAGE,2 L - Tricine- SDS- PAGE和 HPL C进行鉴定 .结果 SDS- PAGE分析表明菌体表达的融合蛋白占菌体总蛋白的 40 0 g· kg- 1 .经 2 L - Tricine- SDS- PAGE分析证实 ,融合蛋白可裂解出 Tα1 单体 ,裂解物经简单的离子色谱可以纯化出 Tα1 单体 ,HPL C鉴定纯化出的 Tα1 纯度可达95 0 g· kg- 1以上 .利用 3H- Td R进行的生物活性测定表明 ,在致有丝分裂原 Con A存在的条件下 ,融合蛋白和 Tα1 单体均具有刺激小鼠脾淋巴细胞分裂增殖的能力 ,与化学合成的Tα1 相比具有相似的生物活性 .结论 该方法是分离纯化Tα1 简便易行 ,经济有效的方法 ;同时也为 Tα1

AIM To purify and prepare thymosin alpha 1 (Tα 1). METHODS The engineering bacteria TOP10 including recombinant plasmid of a fusion expression vector pThioHisA with the tandem Tα 1 gene of 4 repeats (Tα 1④) was used. after bacteria lysated by Bionee Cell Disruoter, the lysate was incubated in the temperature of 80℃ for 10 minutes, and cooled quickly. By Q Sepharose Fast Flow chromatography, the fusion protein was purified from the supernatant of the lysate. At the concentrion of 0.5 mol·L -1 CNBr cleavage of the fusion protein in 700 mL·L -1 formic acid, Tα 1 was purified by ion exchange methods of SP Sepharose Fast Flow and Q Sepharose Fast Flow. These were identified by SDS PAGE, 2L Tricine SDS PAGE and HPLC. RESULTS SDS PAGE analysis showed an induced expression fusion protein band which consititued 400 g·kg -1 of the total bacterial proteins. After CNBr cleavage of the fusion protein, Tα 1 was obtained by ion exchange methods and proved by 2L Tricine SDS PAGE analysis. HPLC showed the purity of Tα 1 was 950 g·kg -1 . Their biological activity was analysed by 3H TdR incorparation. They results showed that the fusion protein and Tα 1 had the similar biological activity to that of the synthesized Tα 1. They could increase the proliferative response of the mitogen ConA stimulated spleen lymphocytes. CONCLUSION Method is convenient and simple for purification and preparation of Tα 1 in large scale.