摘要
目的构建重组人源性纤溶酶原Kringle 5(rhK5)杆状病毒表达载体并进行真核表达。方法采用bac-to-bac杆状病毒表达系统制备杆状病毒Bacmid-rhK5,感染草地贪夜蛾卵巢细胞sf21昆虫细胞,经Western blot确定rhK5在sf21细胞中的表达方式和表达量。用血管内皮细胞抑制实验检测纯化蛋白活性。结果杆状病毒表达载体pFastBac HTB-rhK5构建成功,Western blot检测发现,rhK5在sf21细胞中呈胞内表达,纯化后重组蛋白的产量约为90μg/L。血管内皮细胞抑制实验显示,半数有效剂量(ED50)为4μg/mL。结论rhK5杆状病毒表达载体于悬浮培养的sf21细胞中高效表达,为大量表达更接近天然的具有生物学活性的rhK5蛋白奠定了基础。
Objective To construct baculovirus expression system for the human plasminogen kringle 5（hK5） gene, and detect its expression in Spodoptera frugiperda 21 （ sf21 ）. Methods Baculovirus Bacmid-rhK5 was prepared with bac-tobac baculovirus expression system, and sf21 cells were infected. The expression of rhK5 in sf21 cells was determined by Western blot. The activity of purified protein was detected by vascular endothelial cell inhibition test. Results The pFastBac HTB-rhK5 was successfully constructed, and sf21 cells were transfected with the constructed vector. The output of rhK5 obtained was 90 ug/L, and the protein possessed the inhibitory activity of endothelial cell proliferation. The median effective dose （ EDs0 ） was 4 ug/mL. Conclusion The rhK5 baculovirus expression vector is highly expressed in sf21 cells, which lays a foundation for the the expression of rhK5 protein with biologic activities.
出处
《上海交通大学学报（医学版）》
CAS
CSCD
北大核心
2008年第9期1119-1122,共4页
Journal of Shanghai Jiao tong University:Medical Science
基金
国家自然科学基金(30570524)
上海市科委重点课题(07JC14039)~~
