摘要
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 .
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.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2001年第3期344-349,共6页
Chinese Journal of Biochemistry and Molecular Biology
关键词
胸腺素Α1
基因克隆
融合表达
蛋白质纯化
生物活性
thymosin alpha 1,gene expression,fusion expression,protein purification,biological activity
