Study On Cloning And Expression Of Active Domain Of Rankl And Its Antiserum Preparation In Chicken

RANKL (receptor activator of NF-KB ligand) was firstly found in mammalian, is a member of the tumor necrosis factor (TNF) family. RANKL promotes osteclast differentiation, activation, survival and stimulation and is the terminal factor to interfere the action of osteoclasrs. Considering that RANKL played a very important role in bone metabolism, there were two aims of this study:one was to find a way to obtain functional RANKL protein, the other was to provide a new solution to find the mechanism of cage layer osteoporosis.To obtain the active domain genes of chicken RANKL (chRANKL) (Genbank submission number, EF379383) by PCR method. The chRANKL DNA fragment was cloned into pMD18-T vector. DNA sequencing, restriction enzyme digestion and PCR amplification all confirmed the inserted fragment was a active domain of chicken RANKL gene, which was identical to the chicken RANKL gene published in the Genbank. A 651bp length gene was successfully cloned. The active domain gene was cloned that encoding chicken RANKL mature protein. The RANKL gene was inserted to the expression plasmid pET-28a(+) and expressed in Escherichia coli. BL21(DE3) with IPTG inducement. The SDS-PAGE result showed that the cloned recombinant protein expressed in the form of inclusion bodies in BL21(DE3) with molecular weight of 27KD, and western blot indicated that the expressed protein had satisfied immunobioligical activity. The purified fusion protein was used as antigen to prepare polyclonal antiserum in New Zealand rabbitts. The expressed recombinant protein was immunogen which prepared immune serum by designing a good immune procedures. The antiserum was extracted after four injections of antigen. The results of indirect ELISA and Western blot proved that rabbit polyclonal antiserum obtained was a new reagent with high titration and specificity. Through detection of the chicken osteoblasts and the tissues of osteoepiphysis showe that prepared rabbit anti-chRANKL polyclonal antiserum can be used to identify chicken RANKL protein, and the antibody gas good specificity.