小鼠核因子κB受体活化因子配基活性区的克隆、表达及生物活性分析

Cloning,Expression and Bioactivity Analysis of The Active Domain of The Mouse Receptor Activator of NF-κB Ligand

核因子κB受体活化因子配基是诱导破骨细胞分化、成熟的关键因子,在生物医学研究中应用广泛。本实验以小鼠的骨髓细胞cDNA为模板,采用PCR技术获得小鼠核因子κB受体活化因子配基活性区基因,并将该基因克隆至His标签的融合蛋白表达载体pET28a(+),经鉴定正确的质粒转化至BL21表达菌株中。通过调节诱导目的蛋白表达的培养温度、IPTG浓度及诱导时间,筛选出重组融合蛋白表达的最佳条件。将纯化后的重组蛋白稀释成不同浓度,刺激小鼠破骨前体细胞Raw264.7细胞分化,经抗酒石酸酸性磷酸酶染色后可见破骨细胞的形成,表明该重组蛋白具有较好的生物活性,可替代商品化的鼠源核因子κB受体活化因子配基。

Receptor activator of NF-κB ligand plays a key role in inducing osteoclast differentiation and maturation.It is widely used in biomedical researchs.Mouse＇s marrow cell cDNA was as a template and receptor activator of NF-κB ligand gene was obtained using the PCR technology in the experiment.Then,the gene was cloned with a His tagged fusion protein expression vector pET28a（＋）.The plasmid which was correct through digested identification was transformed into BL21 Escherichia.coli strain.In order to select the optimum condition for fusion protein,the target protein was expressed at the different temperatures,the IPTG concentrations and the induction times.The purified recombinant protein was diluted to different concentrations and was used to stimulate Raw264.7 cell differentiation.The obvious osteoclasts were observed through tartrate-resistant acid phosphatase staining,which indicated that the reorganization protein has good biological activity and could substitute the commercialized mouse receptor activator of NF-κB ligand.