胸腺素α_1 基因的克隆表达及其生物活性

 胸腺素α1(thymosinalpha 1 ,Tα1)作为一种免疫增强剂 ,临床用途广泛 .为大量制备Tα1,按大肠杆菌惯用密码子合成Tα1基因 ,克隆于质粒pUC1 9的EcoRⅠ和PstⅠ位点 .经测序证明序列正确后 ,串联为 4串体 (Tα1④ ) ,经再次测序确认后克隆入pThioHisA的EcoRⅠ和PstⅠ位点 .转化大肠杆菌T0P1 0 ,酶切鉴定正确后 ,经 1mmol LIPTG诱导 4h ,获得硫氧还蛋白与Tα1④的融合表达 ,用离子交换层析纯化融合蛋白 .溴化氰裂解融合蛋白 ,释放出Tα1单体 ,经离子交换色谱纯化出Tα1.采用3 H TdR参入法进行生物活性测定 ,证实融合蛋白和Tα1均具有刺激小鼠脾淋巴细胞分裂增殖的能力 .

Cloning,Expression of Thymosin Alpha 1 Gene in E.coli and Its Purification and Characterization

Thymosin alpha 1(Tα 1)is a immunopotentiating agent widely used in clinic.In order to prepare Tα 1 in large scale,the gene of Tα 1 was synthesized according to the preferential codons of E.coli ,cloned into the Eco RⅠ and Pst Ⅰ sites of pUC19 and sequenced.The tandem Tα 1 gene of 4 repeats was concatenated,and identified by Eco RⅠ and Pst Ⅰ and finally sequenced again.The 4 repeats gene was inserted into Eco RⅠ and Pst Ⅰ sites of thioredoxin fusion expression vector pThioHisA,and the recombinant plasmid was transformed into E.coli TOP10.SDS\|PAGE and densitometry analyses showed the expressed fusion protein,with a molecular weight of about 38kD,which was about 40% of the total bacterial protein after induction at 37℃ by 1 mmol/L IPTG for 4 hours.The fusion protein was isolated and purified by ion exchange chromatography.After CNBr cleavage of the fusion protein in 70% formic acid,Tα 1 was obtained by ion exchange methods and proved by 2L\|Tricine\|SDS\|PAGE analysis.Their biological activity was analysed by 3H\|TdR incorparation.The results showed that the fusion protein and Tα 1 have the similar biological activity to that of the synthesized Tα 1.They could increase the proliferative respones of the mitogen ConA stimulated mice spleen lymphocytes.