摘要
目的为降低CNBr裂解融合蛋白的毒副作用 ,改进利用基因重组技术制备胸腺素α1 (Tα1 )的流程工艺。方法按大肠杆菌惯用密码合成Tα1 基因 ,克隆于质粒pGEM 3Zf的EcoRⅠ和PstⅠ位点 ,裂解位点为羟胺裂解位点Asn Gly对应的核苷酸序列 ,测序正确后用BamHⅠ和BglⅡ位点串联为Tα1 4串体 (Tα1 ④ )和Tα1 8串体 (Tα1 ⑧ ) ,经再次测序确认后克隆入pThioHisA的EcoRⅠ和PstⅠ位点 ,转化大肠杆菌TOP1 0。结果经 1mmol LIPTG诱导4h获得了硫氧还蛋白 (thioredoxin)与Tα1 ④和Tα1 ⑧的融合表达 ,用离子交换色谱可纯化出融合蛋白 ,羟胺裂解融合蛋白 ,能够释放出Tα1 单体。结论羟胺能够裂解融合蛋白释放出天然Tα1 。
Purpose To decrease side effect in the process of cleavage of fusion protein using CNBr and to imporve the preparation process of thymosin alpha 1(Tα 1) using gene recombinant technology.MethodsTα 1 gene was synthesized according to the preferential coden of E.coli , cloned into the EcoRI and PstI sites of pGEM 3Zf and sequenced. The tandem Tα 1 gene of 4 repeats (or 8 repeats) was concatenated, and identified by EcoRI and PstI and finally sequenced again, the 4 repeats gene was inserted into EcoRI and PstI site of thioredoxin fusion expression vector pThioHisA, and the recombinant plasmid was transformed into E.coli TOP10.ResultsSDS PAGE analyses showed the expressed fusion protein, with a molecular weight of about 38 kD, which was about 40% of the total bacterial protein after induction at 37℃ by 1 mmol/L IPTG for 4 h. The fusion protein was isolated and purified by ion exchange chromatography. Tα 1 was obtained from NH 2OH·HCl cleavage of the fusion protein and proved by 2L Tricine SDS PAGE analysis. ConclusionAfter NH 2OH·HCl cleavage of the fusion protein, natural Tα 1 could be obtained by purification process.
出处
《中国生化药物杂志》
CAS
CSCD
2003年第2期55-57,75,共4页
Chinese Journal of Biochemical Pharmaceutics
关键词
胸腺素Α1
融合表达
化学裂解
羟胺
基因克隆
thymosin alpha 1
fusion expression
chemical cleavge
hydroxylamine
gene cloning
