摘要
目的:在大肠杆菌中表达硫氧还蛋白-线虫抗凝血蛋白c2(Trx-NAPc2)融合蛋白,并检测其抗凝活性。方法:将扩增的NAPc2基因经BamHⅠ和HindⅢ双酶切后连接到表达载体pET-32a中,转化至大肠杆菌BL21(DE3),分别经IPTG和乳糖诱导表达;表达产物经镍琼脂糖凝胶FF纯化后,用体外凝血酶原时间(PT)和活化部分凝血活酶时间(aPTT)试验检测抗凝血活性。结果:构建了pET-32a/NAPc2表达质粒,并在大肠杆菌BL21(DE3)中高效表达,表达产物主要以可溶形式存在,纯化的Trx-NAPc2融合蛋白能明显延长PT及aPTT。结论:在大肠杆菌中高效表达了具有生物活性的Trx-NAPc2融合蛋白,为进一步研究NAPc2的功能及应用奠定了基础。
Objective： To express thioredoxin（Trx）-nematode anticoagulant protein c2（NAPc2） fusion protein in Escherichia coli, and to investigate its anticoagulant activity. Methods： PCR products of NAPc2 gene digested by BamH I and Hind lll was ligated into pET32a vector, and the recombinant plasmid of pET-32a/NAPc2 was transferred into E.coli BL21 （DE3）. The expressed Trx-NAPc2 fusion protein induced by IPTG and lactose respectively was purified with His-bind affinity chromatography, and its anticoagulant activity was detected by activated partial thromboblastin time（aPIT） and prothrombin time（PT） assay in vitro. Results： The Trx-NAPc2 fusion protein was expressed and existed mostly as soluble form. The purled fusion protein showed effectively anticoagulant activity in vitro. Conclusion： Trx-NAPc2 fusion protein was expressed in E.coli with high efficiency, and it have effectively bioactivity.
出处
《生物技术通讯》
CAS
2007年第4期578-580,共3页
Letters in Biotechnology
基金
广东省自然科学基金项目(04011381)
关键词
线虫抗凝蛋白c2
融合蛋白
表达
抗凝活性
nematode anticoagulant protein c2
fusion protein
expression
anticoagulant activity
