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目的:在大肠杆菌内表达基因重组人血管生长素衍生物Asp116His(rhD116H-ANG)并恢复其天然活性。方法:采用EcoR和BamH双酶切,重组构建pBV220/D116H-ANG表达载体,转化大肠杆菌TG1基因工程菌,温控表达,破菌提取包涵体,变性、溶解、纯化、复性、浓缩,以鸡胚绒毛尿囊膜法及RNA降解试验测活。结果:成功表达rhD116H-ANG,表达量约30%,经纯化、复性、测活,证实获得活性蛋白,并取得较高得率及复性效率。结论:本实验建立的原核表达体系是获取活性rhD116H-ANG的有效途径,为规模化制备奠定了技术基础。
OBJECTIVE: To express recombinant human angiogenin Asp116His (rhD116H-ANG) and restore its natural activity in E. coli. Methods: The recombinant plasmid pBV220 / D116H-ANG was constructed by digestion with EcoR and BamH. The recombinant plasmid was transformed into E. coli TG1. The recombinant plasmid was transformed into E.coli TG1. Chick embryo chorioallantoic membrane assay and RNA degradation test. Results: The rhD116H-ANG was successfully expressed in about 30% of the cells. After purified, refolded and measured, rhD116H-ANG was confirmed to obtain the active protein with high yield and refolding efficiency. Conclusion: The prokaryotic expression system established in this experiment is an effective way to obtain active rhD116H-ANG, which lays the technical foundation for the large-scale preparation.