Studies On Cloning, Expression And Biological Activities Of Chicken RANKL

RANKL (receptor activator of NF-κB 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 osteoclasts. 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.ExperimentⅠCloning and sequencing analysis of encoding genes in chicken ligand of receptor activator of NF-KbTo obtain the encoding gene of chicken RANKL (chRANKL) (GenBank submission number, EF379383) from the chicken embryo osteoblasts by RT-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 complete chicken RANKL gene with ORF, which had the identities of 99.3% with the chicken RANKL gene published in the GenBank. A 1203 bp length gene was successfully cloned. The sequence reported here had 55.4%,52% and 55.7% identity to human, mouse and dog, respectively. The amino acid sequence phylogenetic result showed that RANKL gene had high conservation in evolution. The results supply the basis to do research in cage layer osteoporosis.ExperimentⅡProkaryotic expression and purification of recombinant chRANKL proteinA pair of primers were designed to clone the gene encoding chicken RANKL mature protein, then the RANKLm gene was inserted to the expression plasmid pET-32a(+) 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 64 kD, and western blot indicated that the expressed protein had satisfied immunobiological activity.ExperimentⅢOsteoclast formation induced by chRANKL from chicken embryo marrow cellsThe aim of this experiment was to find a simple and effective inducing culture system of the osteoclast-like cells from the embryonic chicken in order to supply a new way to study the mechanism of the cage layer osteoporosis. Tibias and humeri were excised from chicken embryos (18 days of age) and cleaned of extraneous soft tissue. The bone marrow was washed with a-MEM using injector. The cell supematants obtained were inoculated on culture dishs. The nonadherent cells were collected after two-hour and resuspend to make the cells be 2x106/mL. Then cell supernatants inoculated again, during which chRANKL and M-CSF were added into wells at different concentrations. Osteoclasts-like cells were successfully obtained with features of multinucleated giant size, positive staining for TRAP and bone absorptive lacunae on the bone slice. The results showed that chRANKL could induce preOCs to form OLCs, at meanwhile, chOPG could interrupt the function of chRANKL’s effect on OLC formation.Experiment IV Expression of chRANKL in methylotrophic yeast Pichia pastoris and the study on bioactivity of recombinant proteinAnother pair of primers were designed to clone the gene encoding chicken RANKL mature protein, then the chRANKLm’was inserted into vector pPICZa-A. The constructed plasmid was transformed into yeast X-33 by electroporation. The recombinant transformants were selected by Zeocin. Induced by the addition of methanol every 24 hours, the product analyzed by SDS-PAGE was sized about 54 kD at a yield of 220 mg per litter of culture. The result of Western Blot indicated that the recombinant protein could react with anti-human RANKL antibody and the expressed protein had specific antigenicity.Experiment V Effect of chRANKL frozen product expressed in methylotrophic yeast Pichia pastoris on mature osteoclast in vitro and vivo50 ISA cage layers were divided into 5 groups. Group A is control, chRANKL were injected to B, C, D, E groups at the dosage of 0.01 mg/kg,0.05 mg/kg,0.1 mg/kg,0.5 mg/kg. After 2 hours, blood was collected to check the concentration of Ca++. We also add different concentration of 2 ng/mL,6 ng/mL and 10 ng/mL of RANKL into mature osteoclast. After 3 d, we observe the change of bone resorption lacunae and the change of Ca++ in the culture medium. We found that the chRANKL could stimulate the activity of mature osteoclast in dose-dependent manner and increase the concentration of Ca++ in vitro and vivo. The study demonstrated that the chRANKL also can stimulate the activity of mature chicken osteoclast.