【摘要】 以化学合成的H iru log 18基因为模板,经PCR扩增、限制性内切酶Xho I和Kpn I消化,将编码H iru-log 18的基因片断插入表达载体pET-32b中编码硫氧化还原蛋白(Trx)基因序列的下游.用重组质粒转化感受态大肠杆菌-BL21(DE3),并用IPTG诱导表达Trx-H iru log 18融合蛋白.复性、肠激酶酶切、N i-Sepharose和HPLC纯化后,用质谱测定其分子量并进行活性测定.检测结果表明构建的PET-32b/H iru log 18能够在大肠杆菌中高效表达,表达的H iru log 18具有较低的凝血酶抑制活性并能够竞争抑制Angiomax活性.更多还原

【Abstract】 Hirulog 18 DNA fragment chemically synthesized is amplified with PCR and digested with restriction endonuclease: Xho I and Kpn I,and inserted into the prokaryotic expression vector pET 32b.The host cell strain BL21(DE3) transformed with recombinant plasmid Hirulog 18/Trx is induced at 37OC to express the recombinant product.Having been renaturalized,the fusing protein product is digested with enterokinase,purified by Ni-sepharose and HPLC.Hirulog 18 gene product is identified by MS.The activity assay is also performed.These results show the designed plasmid PET-32b/ Hirulog 18 can be expressed successfully in E.coli,and the gene product has a weak inhibitory activity against thrombin and competes with Angiomax.更多还原