摘要
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 derivative Asp116His (rhD116H ANG) in E. coli and converse the product to its active form. Methods: A prokaryotic expression vector named pBV220 was used to express the rhD116H ANG gene by inserting the EcoRⅠ and BamHⅠ digested gene into the same site of the expression vector. Propagated in E. coli strain TG1, the gene produced a recombinant protein (MW 14 400 ±) in form of inclusion bodies after 42℃ thermo induction. The inclusion bodies were extracted and solubilized in a degenerating chaotropic agent solution, and then subjected to purification, renaturation and concentration steps. The product was assayed by CAM methods and RNA degradation test for evaluation of its biological activity. Results: rhD116H ANG was successfully expressed in this system with a yield of about 30%. The steps that ensured a fully active protein, which was confirmed by its angiogenic and RNase like activity, with high yielding rate and renaturation efficiency. Conclusion: This prokayotic expression system is competent for obtaining fully active rhD116H ANG protein and lays a solid foundation for the magnitudinal preparation of the protein.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
1999年第12期937-940,F004,共5页
Academic Journal of Second Military Medical University
基金
国家自然科学基金!批准号 3 9970 73 5
