重组胸腺素α1的纯化及活性

Purification of Recombinant Thymosin Alpha 1 in Escherichia coli and Its Activity

将胸腺素α1基因 4串体克隆于 pThioHisA融合表达载体 (pThioHisA Tα1④ ) ,转化大肠杆菌TOP10。在IPTG诱导下 ,融合蛋白得到了高效表达。SDS PAGE分析确定诱导表达的融合蛋白占菌体总蛋白的 40 % ,且表达的融合蛋白主要以可溶形式存在。把用离子交换层析纯化出的目的蛋白进行CNBr化学裂解 ,经简单的离子交换层析可纯化出胸腺素α1单体 ,HPLC鉴定胸腺素α1的纯度达 98%。利用3 H TdR进行生物活性测定 ,证实融合蛋白和胸腺素α1单体均具有在致有丝分裂原ConA存在的条件下 ,刺激小鼠脾淋巴细胞分裂增殖的能力 ,与化学合成的胸腺素α1相比具有相似的生物活性。

The tandem thymosin α1 gene of 4 repeats was constituded and was cloned into a fusion expression vector pThioHisA(pThioHisA\|Tα1④).After transforming into TOP10,Higher levels of expression of fusion protein,with a molecular weight of about 38kD,was induced at 37℃ by 1 mmol/L IPTG for 4 hours.SDS PAGE analysis showed an induced expression products band which consititued 40% of the total bacterial proteins.The fusion protein was isolated and purfied by simple ion exchange chromatography.After CNBr cleavage of the fusion protein in 70% formic acid,thymosin α1 was obtained by the ion exchange methods of SP\|Sepharose Fast Flow and Q\|Sepharose Fast Flow and proved by 2L\|Tricine\|SDS\|PAGE analysis.HPLC showed the purity of thymosin α1 was 98%.Furthermore,their biological activity was analysed by 3H\|TdR incorparation.The results showed that the fusion protein and thymosin α1 have the similar biological activity to that of the synthetic thymosin α1.They could increase the proliferative respones of the mitogen ConA stimulated spleen lymphocytes.This method provided a reference method in preparation thymosin α 1 in large scale.