抗栓肽和尿激酶原（B链）嵌合体的构建与表达

北京大学学报（自然科学版）

抗栓肽和尿激酶原（B链）嵌合体的构建与表达

张磊亮, 管胜昔, 姜靓靓, 俞梅敏, 茹炳根

北京大学生命科学学院，蛋白质工程国家重点实验室，北京，100871

收稿日期:2003-04-09 出版日期:2004-07-20 发布日期:2004-07-20

Construction and Expression of the Recombinant Dscu-PA(B)

ZHANG Leiliang, GUAN Shengxi, JIANG Jingjing, YU Meimin, RU Binggen

College of Life Sciences, National Lab of Protein Engineering, Peking University, Beijing, 100871

Received:2003-04-09 Online:2004-07-20 Published:2004-07-20

摘要/Abstract

摘要： 通过基因工程重组技术，将抗栓肽（decorsin）基因与尿激酶的B链即scu-PA(B)基因用柔性Linker((Gly₄Ser)₃)融合在一起，构建新的嵌合体基因dscuPA(B)，在大肠杆菌Rosetta(DE3)plysS中通过IPTG诱导表达, 并考察了重组质粒在Rosetta(DE3)plysS在传代中的分离稳定性。该融合蛋白在大肠杆菌中是以包涵体的形式存在，对包涵体进行变性及复性后，并通过Zn²⁺螯合柱和SP阳离子交换柱进行纯化。质谱分析表明分子量为33.735kD，与理论值相符。目的蛋白质的纯度可达90%。纤维蛋白平板法测得嵌合分子的比活为90000IU/mg，与scuPA-32k的比活相近。体外血小板聚集实验表明融合蛋白有较强的抑制血小板聚集的功能，抑制常数IC50为0.31μmol/L。以上这些结果表明，该融合蛋白不但具有较强的溶栓功能，而且具有抗栓功能，可能是一种具有很好发展前景的双功能溶栓抗栓药物。

关键词: 尿激酶原, 抗栓肽, 质粒稳定性, 血小板聚集

Abstract: A recombinant chimeric plasminogen activator (dscu-PA(B)) was constructed, consisting of the decorsin (platelet aggregation inhibitor), fused via a 15-amino-acid linker (Gly₄Ser)₃ sequence to the N-terminal of the B chain of urokinase (comprising Leu159 through Leu 411). The recombinant protein was produced in E.coli host strain Rosetta(DE3)plysS after IPTG induction and exited in inclusion body. The stability of plasmid was studied. After refolded in vitro, the chimeric protein was purified by Zinc chelate-Sepharose chromatography and SP Sepharose chromatography in sequence. The molecular weight was 33.735kD by MALDI-TOF analysis. The special activity of the chimera was 90000IU/mg detected by fibrin plate determination. It was also shown that chimera inhibited ADP-induced platelet aggregation in a concentration dependent manner. Inhibition of approximately 50% aggregation was achieved at a concentration of approximate 0.31μmol/L, which was a little lower inhibition potential than that of decorsin. These results showed that the chimeric protein had not only high thrombolytic activity but also anti-thrombus function. Further evaluation of the thrombolytic potential in appropriate animal models seems to be investigated.

Key words: urokinase, decorsin, stability of plasmid, platelet aggregation

中图分类号:

张磊亮, 管胜昔, 姜靓靓, 俞梅敏, 茹炳根 . 抗栓肽和尿激酶原（B链）嵌合体的构建与表达[J]. 北京大学学报（自然科学版）.

ZHANG Leiliang,GUAN Shengxi,JIANG Jingjing,YU Meimin,RU Binggen. Construction and Expression of the Recombinant Dscu-PA(B)[J]. Acta Scientiarum Naturalium Universitatis Pekinensis.

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https://xbna.pku.edu.cn/CN/Y2004/V40/I4/588