Cloning, Expression And Bioactivity Analysis The Active Domain Of Mouse Receptor Activator Of NF-κB Ligand (mRANKL)

Receptor activator of NF-κB ligand (RANKL), a key factor in inducing osteoclast differentiation and maturation, is widely used in biological and medical research. RANKL mainly binds RANK to activate RANKL-RANK signaling. Once RANK activated, some downstream pathways, such as:NF-κB pathways, P38 pathways, ERK pathways, JNK pathways would start. These signaling pathways can induce osteoclast differentiation, maturation and survival. As RANKL plays an important role in osteclast development, its depletion would cause a variety of disorders of bone diseases, including osteoporosis, rheumatoid arthritis, etc. A variety of drugs which are associated with RANKL and RANKL-RANK signaling have been explored to treat skeleton disease caused by RANKL. So it is very significant to make highly pure mRANKL protein for inducing pre-osteoclast differentiation. This platform not only provide fundamental research model for drug screening which aim to bone diseases treatment, but also cutting down the cost of lab.In our experiment, the DNA sequence encoding the active domain of mouse's receptor activator of NF-κB(RANKL) was amplified from bone marrow cell by polymerase chain reaction(PCR). Then the PCR production were cloned into the expressing plasmid pET28a(+) with His-tag. The recombinant plasmid pET28a-mRANKL transformed Escherichia BL21 subsequently. Found out the best conditions for recombinant protein of mRANKL by changing the experiment condition including temperature,the concentration of IPTG and induction time. A large amount of recombinant protein of mRANKL was prepared according to the optimal condition, filtered and diluted into different concentration. The recombinant proteins of mRANKL and mRANKL purchased respectively were added to monocytes/macrophages Raw264.7 cell medium. Obviously, osteoclaste was found in 3.5 day. The result indicates that the recombinant protein mRANKL possesses biological activity, which can replace the commercial mRANKL.