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| The cloning, expression and bioactivity analysis of chRANKL |
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Abstract To clone chicken RANKL(chRANKL) gene by RT-PCR method and obtain the chRANKL protein expressed in E.coli and analyse its bioactivity. The encoding sequence of chicken RANKL was amplified from embryo chicken osteoblasts total RNA by RT-PCR. The PCR product was inserted into the prokaryotical expression vector pET-32a(+). The recombinant vector was transformed into E.coli BL21(DE3)Plys and IPTG inducement. Then the purified protein was added to coculture with the mature chicken osteoclasts in vitro to observe its activation. Result showed that the PCR product was about 1200 bp long, which was consistent with the expected one. The sequence of insert corresponded with the published encoding sequence of chicken RANKL. Plasmid pET32a(+)-chRANKL was transformed into E.coli BL21(DE3)Plys and a 64 Kd protein was expressed by IPTG inducement, and Western blotting indicated that recombinant protein had good antigenicity. We also found that the chRANKL could stimulate the activity of mature osteoclast which increased both the number and the area of bone resorption lacunae relative to dose-dependent manner.
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Received: 19 July 2007
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