CAJ | 학술논문

为开发一种新的有临床应用价值的抗血栓药物,根据水蛭素保持抗凝活性的20肽片段,设计并构建了水蛭素相关肽(Hi-lys)与天冬酰胺酶C端的融合表达系统.为方便目的肽与融合伙伴的分离,增加了富含带电序列的8肽(KRKRKKSR)及酸敏感的天冬氨酰-脯氨酸(Asp-Pro)位点,获得了表达质粒pED-P8-Hi-lys.将其转化E.coli BL-21,玉米浆培养基(kanr)培养,乳糖诱导获得融合蛋白(AnsB-C-P8-Hi-lys)的高效表达.通过细菌裂解、包涵体洗涤、尿素溶解、乙醇沉淀、酸水解和DEAE-纤维素52柱层析纯化获得目的肽Hi-lys,用凝血酶测定法测得其抗凝活性为50 ATU/mg.
위개발일충신적유림상응용개치적항혈전약물,근거수질소보지항응활성적20태편단,설계병구건료수질소상관태(Hi-lys)여천동선알매C단적융합표체계통.위방편목적태여융합화반적분리,증가료부함대전서렬적8태(KRKRKKSR)급산민감적천동안선-포안산(Asp-Pro)위점,획득료표체질립pED-P8-Hi-lys.장기전화E.coli BL-21,옥미장배양기(kanr)배양,유당유도획득융합단백(AnsB-C-P8-Hi-lys)적고효표체.통과세균렬해、포함체세조、뇨소용해、을순침정、산수해화DEAE-섬유소52주층석순화획득목적태Hi-lys,용응혈매측정법측득기항응활성위50 ATU/mg.
A new fusion expression vector, pED-P8-Hi-lys was designed and constructed. It includes four parts, a 20 peptide sequence of hirudin that can maintain anticoagulant activity, the C-terminus of asparaginase as a fusion partner, basic octopeptide (KRKRKKSR) that makes the fusion partner easy to remove, and the unique acid-labile aspartyl-prolyl bond. It was transformed into E. coli BL-21 and the fusion protein (AnsB-C-P8-Hi-lys) was expressed effectively as inclusion bodies after inducing by lactose. The objective peptide Hi-lys was purified by means of cell disruption, washing, ethanol precipitation, acid hydrolysis, and DEAE-cellulose 52 column chromatography. The antithrombin activity of the purified Hi-lys peptide was about 50 ATU/mg by thrombin activity assays.