OPG-HSP65重组蛋白的原核表达及其生物学活性研究  

Expression and Biological Characterization of Human Osteoprotegerin Fused with Mycobacteria Heat Shock Protein 65

作　　者：张月[1] 刘舒[1] 马静[1] 李慎涛[1] 王炜[1] 张权庚[1] 赵文明[1] 

机构地区：[1]首都医科大学基础医学院免疫学系,北京100069

出　　处：《中国生物工程杂志》2009年第4期22-27,共6页China Biotechnology

基　　金：国家自然科学基金资助项目(30873185);北京市教委科技发展计划项目资助(KM200810025013)

摘　　要：PCR扩增OPG-HSP65基因,构建原核重组表达载体pET-28a-OPG-HSP65,转化大肠杆菌BL21(DE3),经IPTG诱导表达产生包涵体形式的目的蛋白。对重组蛋白进行Western blot检测表明,重组蛋白能与抗His-Tag单克隆抗体及鼠抗人OPG单克隆抗体特异性结合。对重组蛋白进行尿素洗涤纯化,进而透析、复性。经破骨细胞生长抑制实验和抑炎实验表明,重组蛋白能减少破骨细胞生成及减轻迟发型超敏反应小鼠模型炎症反应。A fused functional gene of human OPG and Mhsp65 was amplified by PCR, and cloned into the prokaryotic expression vector pET-28a. The BL21 （DE3） strain of E. coli was transformed using the recombinant plasmid pET-28a-OPG-HSP65 and the expected protein was expressed by induction with IPTG. Result of SDS- PAGE indicated that the expected recombinant protein of 23 kDa was expressed with high yield as inclusion body. The fusion protein could be specifically recognized by both the anti-His antibody and anti-human OPG monoclonal antibody in Western blot analysis. The purified and refolded fusion protein could inhibit osteoelast proliferation and bone absorption in vitro. The results of mouse ear swelling assay and expressions of TNF-α, IFN-γ and IL-17 mRNAs detected by real-time quantitative PCR demonstrated that the fusion protein had an anti-inflammation activity. The results suggest that the fusion protein of human OPG and Mhsp65 may act as a potential therapeutics for rheumatoid arthritis.

关 键 词：骨保护素 热休克蛋白65 重组蛋白 骨保护作用 抑炎作用 

分 类 号：Q81[生物学—生物工程]