小鼠骨保护素配基胞外片段的表达、纯化及生物活性分析

骨保护素配基(OPGL)是调节破骨细胞分化和成熟的核心细胞因子。由小鼠骨组织提取总RNA，RTPCR扩增得到小鼠OPGL胞外片段(sOPGL)cDNA，以特定策略克隆人表达载体pET-42a( )，以便使未来表达产物的融合标签序列能够完全被因子Xa切除。重组载体在大肠杆菌中诱导表达可获得高水平的47kD产物，Western blotting证实它可被OPGL抗体识别。经Glutathione-sepharose 4B亲和层析，除融合蛋白外，还有一约30 kD蛋白与层析柱发生了特异性亲和。该30kD蛋白可被GST-IGF-I多克隆抗体识别，但不能被OPGL抗体识别，提示它的产生乃由于融合蛋白在融合位点附近发生裂解。融合蛋白经Xa因子裂解和进一步纯化，得到分子量约17．5kD的sOPGL。生物活性分析证明，重组sOPGL可以促进OLC的生成，并呈现剂量依赖关系。

Expression,Purification and Bioactivity Characterization of Extracellular Domain of Murine Osteoprotegerin Ligand

Osteoprotegerin ligand (OPGL) is a key regulator of formation and activation of osteoclasts.In the present study,the cDNA encoding the extracellular domain of murine OPGL (sOPGL) was synthesized by RT-PCR and cloned into fusion expression vector pET-42a( ) in a certain strategy on purpose that the fusion tag could be completely removed by factor Xa from the expressed fusion protein without any vector-encoded sequence left.Induced with IPTG,the recombinant E.Coli cells produced a 47 kD protein in high level that could be recognized,through Western blotting analysis,by the antibody against OPGL.The expressed products were purified through Glutathione-sepharose 4B affinity chromatography.Along with the fusion molecule,a protein about 30 kD was also specifically bound to the resin.The 30 kD molecule could be recognized by polyclonal antibody against GST-IGF-1,but not by antibody against OPGL.It suggested that the 30 kD molecule was derived from the degradation of the fusion protein.After the cleavage with factor Xa and further purification,the fusion tag was removed and the recombinant sOPGL was obtained.Finally,we confirmed that the recombinant sOPGL could promote osteoclast formation from mouse bone marrow cells in a dose dependent manner.