Cloning, Recombinant Expression And Elementary Biological Activity Of Human Osteoclastogenesis Inhibitory Factor/Osteoprotegerin

Using human liver cell totol RNA as a template, human Osteoclastogenesis inhibitory factor (OCDF)/ Osteoprotegerin (OPG) mature peptide gene was amplified by reverse transcription PCR and cloned into pMD18-T vector and sequenced. The sequence of obtained human OdFm clone was completely identical to that of OCIFm in GenBank. After digested with appropriate restriction enzyme, the gene encoding OCIF/OPG mature peptide (OCIFm) was inserted into prokaryotic expression vector pProEX-HTa directively and transformed into E.coli TB1 for expression via inducing by DPTG 6His-OCIF fusion protein was identified by SDS-PAGE and Western blot analysis. SDS-PAGE and Western blot analysis proved that the relative molecular weight of the expressed product was about 80kD, which was consistent with the expection. Fusion protein accounted for about 20% of total bacteria protein and could react specifically with anti-human OCIF monoclone antibody. Human OCIF/OPG mature peptide gene was successful cloned and expressed in E.coli. The bioactivity of expression product was observed in vitro.Using a two hybrid system of Stratagene BacterioMatch, a fused expression plasmid was constructed between pTRG and TRAIL'S soluble extracellular domain as the "bait", while the fused expression plasmids were also constructed between pBT and extracellular domain of DR4, DR5 or OPG, respectively, as the "target". The each recombinant pTRG and pBT were co-transformated into bacterioMatch two-hybrid system reporter strain E. coli XL 1-Blue MRF. The protein-protein interactions were detected by its carbenicillin-resistant property and validated by β-galactosidase activation. The results showed that the four groups, including that co-transformed by pTRG-TRAIL and pBT-DR4, pTRG-TRAIL and pBT-DR5, controls (pBT-LGF2 and PTRG-Galll), and pTRG-TRAIL and pBT-OPG grew as carbenicillin-resistant colonies.Three groups showed higher resistance to carbenicillin (above 500|ag/ml) except for the last of the groups (under 250jJ.g/ml). The positive colonies were further validated by (3-galactosidase activation on X-Gal-PETG plates. The others, which were pTRG-TRAIL and pBT-LGF2, pBT-DR4 or pBT-DR5 or pBT-OPG and pTRG-Galll, or non-co-transformed plasmids themselves, were negative on carbenicillin or X-Gal-PETG plates. Thus, the straight interaction between TRAIL and OPG was validated. In addition, bacterial two-hybrid system could be used for analysis and identification of certain extracellular protein-protein interactions. The interaction was also validated between OCTF/OPG and TRAIL by neutralization which recombinant fusion 6His-OCIF inhibited TRAIL-induced cytotoxic effect on cultured SW1990 cell line.In the present studies, the mature OCEF/OPG code sequence was cloned, and fusion protein recombinanted in E. coli. The interaction was between OCTF/OPG and TRAIL, but their affinity was lower than that between TRAIL and DR4 or DR5. The fusion recombinant 6His-OCTF induced apoptosis cultured osteoclast. and inhibited cytotoxic effect induced by TRAIL on tumor cells. The work founded further study about OCIF/OPG...