胸腺素α_1-硫氧还蛋白融合蛋白基因的表达及其生物学功能的初步研究  

Expression and Biological Activities of Thymosin Alpha1-Thioredoxin Fusion Protein

作　　者：赵永同[1] 石继红[1] 赵宁[1] 王俊楼[1] 颜真[1] 韩苇[1] 张英起[1] 

机构地区：[1]第四军医大学生物技术中心,西安710032

出　　处：《药物生物技术》2001年第2期67-71,共5页Pharmaceutical Biotechnology

摘　　要：胸腺素α1(thymosinalpha 1,Tα1)作为一种免疫增强剂 ,临床用途广泛。为了大量制备Tα1,按大肠杆菌惯用密码子用人工方法合成Tα1基因 ,克隆于 pUC19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TOP10菌种 ,酶切鉴定证明正确后 ,经 1mmol/LIPTG诱导 4h ,获得硫氧还蛋白与Tα1④的融合表达 ,并经Westernblot分析证实。用离子交换柱层析可纯化出硫氧还蛋白与Tα1④的融合蛋白 ,利用3H -TdR掺入法证实 ,该融合蛋白具有刺激小鼠脾淋巴细胞的活性。Thymosin alpha 1 (Tα 1) is a immunopotentiating agent widely used in clinic. In order to prepare the Tα 1 in large scale, the gene of Tα 1 was synthesized according to the preferential codons of E.coli , cloned into the EcoRI and PstI sites of pUC19, sequenced correctly, the tandem Tα 1 gene of 4 repeats was concatatenated, and identified by EcoRI and PstI and finally sequenced again. The 4 repeats gene was inserted into EcoRI and PstI sites of thioredoxin fusion expression vector pThioHisA, and the recombinant plasmid was transformed into E.coli TOP10. By 12% SDS-PAGE and densitometry analysis, higher levels of expression of fusion protein, with a molecular weight of about 38 kD, was about 40% of the total bacteria protein after induction at 37℃ by 1 mmol/L IPTG for 4 hours , and was identified by western blotting. The fusion protein was isolated and purified by DEAE-Sepharose FF and SP-Sepharose FF ion exchange chromatography. Its biological activity was analysed by 3H-TdR incorparation. The results showed that the fusion protein could increase the porliferative responses of the mitogen ConA stimulated mice speen lymphocytes.

关 键 词：胸腺素Α1 硫氧还蛋白 基因克隆 融合表达 蛋白质 纯化 生物活性 细菌 

分 类 号：Q813.6[生物学—生物工程] R979.5[医药卫生—药品]