Studies On Clone And Expression And Biological Activities Of Chicken Osteoprotegerin

Osteoprotegerin(OPG) was firstly found in mammalian, it was a secreted protein which could inhibit the differentiation of osteoclast precursor cells and the activity of mature osteoclast, and also induce osteoclast apoptosis. A variety of hormones and cytokines regulated the bone metabolism through the regulation of OPG/RANKL ratio in fact. Considering that OPG had a very important role in bone metabolism, there were two aims of this study, one was to find a way to obtain functional OPG protein, the other aim was to evaluate the possibilisity of gene therapy for cage layer osteoporosis.1. The more simple and effective method of isolating the osteoclastic cells from the embryonic chicken was built in order to supply a new way to study the biological activities of osteoprotegerin as well as the cage layer osteoporosis. Tibias and humeri were isolated from 10 chicken embryos(18 d of age)and cleaned of extraneous soft tissue, split each bone lengthwise and quickly flush the inside surface of bones to abtain the cell supernatants. Cell suspension were replated at 24-well dishes, nonadherent cells were washed off after 2 h. The adherent cells had a classic characteristic of osteoclasts: be multinucleated giant cells; like a fried egg, a dumbbell and a ellipse; staining positively for TRAP in cells; forming bone absorptive lacunae on the bone slices.2. Based on the more effective method of isolating osteoclastic cells from the long bone(tibia and humeri) of embroynic chicken, the effects of ipriflavone and dexamethasone on the bone resorption of osteoclastic cells was observed. After 6-day co-cultured, bovine bone slices were taken out for toluidine blue staining in order to idenitfy the vitality of the cultured cells. The results showed that, ipriflavone at 10-⁷ to 10-⁹ M reduced the number and area of bone resorption pit lacunae(P<0.05), while dexamathesone at 10-⁶ to 10-⁸ M could increase the the number and area of bone resorption pit lacunae. Both effects of these two drugs had a positive correlation with their dosage.3. To abtain the encoding genes of chicken osteoprotegerin(GenBank submission number, DQ098013) from the chicken embryo osteoblasts by RT-PCR method, the chOPG 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 OPG gene with ORF, which had the identities of 99.3% with the chicken OPG gene published in the Genbank. The ammo acid sequence phylogenetic of OPG between chicken and mammalian showed that, the chicken had 69.95% identity to human and 65.51% identity to rat and mouse. The results supplied the basis of the recombinant expression of chOPG4. A pair of primers were designed to sub-clone the gene encoding chicken OPG mature protein, then the OPGm gene was inserted to the expression plasmid pET-32a(+) and expressed in Rosetta-gami(DE3) pLysS with IPTG inducement. The SDS-PAGE result showed that the cloned recombinant protein expressed in the form of inclusion bodies in Rossetta with molecular weight of 63 kD and amounted to 11.9% of the whole protein, and western blotting indicated that the expressed protein had satisfied immunobiological activity. Pure protein was obtained by Ni²⁺-NTA chelating column for preparation of anti-OPG polyclonal antibody by immunizing New Zealand rabbit, the titer of antiserum generated was 1:10240 by ELISA. Western blotting analysis showed that it could bind with OPG protein specially expressed in Pichia Yeast.5. Another pair of primers were designed to sub-clone the gene encoding chicken OPG mature protein, then the chOPGm' 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 43 kD and 53 kD at a yield of 200 mg per litter of culture. The result of Western blotting indicated that the recombinant protein had specific antigenicity mainly owing to heterogeneous glycosylation. The chOPG recombinant protein at certain concentration could restrain the activity of mature osteoclasts in vitro, that was to say, the number and area of the bone resorptive lacunae were decreased.6. 100 ISA cage layers were divided into 5 groups. Group A was controll, chOPG were injected to B, C, D, E groups at the dosage about 100μg, 200μg, 300μg, 400μg per four days. Every week, hen blood was collected to check the Ca, P, AKP. Each day, the number of eggs and abnormal and broken eggs were noted. The thickness and intensity of eggshells were tested every week. At last, bone biomechanical property and bone radiographic density were measured. The total average rate of eggs of group B and C was significantly higher than that of group A and the other groups. The total average rate of abnormal and broken eggs of group B, C, D and E were all decreased, group B and C were significantly lower than group A. The thickness and intensity of eggshells between each group were not significant. The serus level of Ca of addministrated groups were significantly lower than the control while no difference of the serus level of P. The serous level of AKP of group B, C and D were significantly higher than the control; The serous level of BGP of group C and D were also significantly higher than the control; while the serous level of StrACP of group E was significantly lower than the control. The bone biomechanical property and bone radiographic density of group C was significantly higher than the control. The study demonstrated that the appropriate dosage of chOPG may improve bone metablism and the production of cage layer during the late cycle.