人破骨细胞生成抑制因子在真核细胞中的表达

Expression of human osteoclastogenesis inhibitory factor(osteoprotegenin) in eukaryotic cell

目的克隆人破骨细胞生成抑制因子(OPG/OCIF)编码区基因并在真核 细 胞中表达，为进一步研究OPG/OCIF的生理功能及探讨骨质疏松症等疾病的治疗奠定基础。方法以人骨肉瘤细胞系MG63的mRNA为模板，采用RT-PCR法得到人OPG/OCIF 的 编码区cDNA，构建真核表达载体并转染成肌细胞，应用核酸杂交及western blot法证实OP G/OCIF在真核细胞中表达。结果获得人破骨细胞生成抑制因子(OPG/OCI F) 编码区全长cDNA，经序列测定证实与文献报道的OPG/OCIF编码区基因的核苷酸序列完全一致 。成功构建OPG/OCIF真核表达载体pcDNA3-OPG。重组质粒转染小鼠成肌细胞C2C12，建立稳 定转染OPG/OCIF编码区cDNA的细胞系，经核酸杂交及western blot法证实OPG/OCIF在真核细 胞中表达。结论 获得人破骨细胞生成抑制因子(OPG/OCIF)编码区全长cD NA并证实在真核细胞中表达，为OPG/OCIF进一步的功能研究奠定了基础。

Objective To construct eukaryotic expression plasmid of human osteoclastogenesis inhibitory factor(osteoprotegenin) and detect its expression in C2C12 cell lines. Methods Using the isolated total RNA from osteosacoma cell line MG63 stimulated by rhBMP-2 as a template, the cDNA encoding hOPG was amplified by reverse transcription-polymerase chain reaction(RT-PCR) method. The PCR product was cloned into pUC19 and sequenced. Further , the OPG encoding region gene was inserted into eukaryotic expression vector pcDNA3. Fi-nally,the constructed recombinant plasmid pcDNA3-OPG was transformed into C2C12 cell line and OPG expression in cell line C2C12 was determined by RNA blot hybridization and western blot. Results The sequence of OPG /OCIF cDNA obtained in this experiment is the same as that of reported. Recombinant plasmid pcDNA3-OPG was constructed successfully and transformed into C2C12 cell line. OPG over-expression cell line C2C12 was selected and confirmed by RNA blot hybridization and western blot. Conclusion OPG/O CIF cDNA was obtained and its expression in cell line C2C12 was determined in this experiment. These results enable further studies on function and structure of human OPG/OCIF.