Cloning, expression and bioactivity of chicken receptor activator of NF-κB ligand ( chRANKL )

Abstract

Using reverse transcription polymerase chain reaction (RT-PCR) and gene splicing by overlap extension (SOE-PCR), the DNA sequence encoding the chicken's receptor activator of NF -κB ligand (RANKL) was amplified, and then cloned into the expressing plasmid pET32a(+) with His-tag, and was highly expressed in Echerichia coli. Then the purified protein was added in primary cultures of chicken osteopclasts to observe its bioactivity. The results showed that the size of PCR product was 1 200 bp which was consistent with the expected one, and the relative molecular weight of induced protein was 64 kD. Western blot indicated that the induced protein could react with anti-His antibody. It was also found that the induced protein could stimulate mature chicken osteoclasts to resorb bone.