人破骨细胞分化因子基因的克隆及在COS-7细胞中的表达

Cloning of Human osteoclast differentiation factor and its expression in COS-7 cells

目的 克隆人破骨细胞分化因子 (ODF)基因并在真核细胞中表达。方法 以人骨肉瘤细胞系MG6 3的总RNA为模板 ,采用反转录 聚合酶链反应 (RT PCR)法得到ODF的编码区cDNA ,构建真核表达载体pcDNA 3 1(+) ODF ,在脂质体介导下转染非洲绿猴肾细胞系 (COS) 7,经G4 18压力筛选建立稳定转染人ODF的细胞系 ,Western印迹检测其在COS 7细胞中的表达。结果 获得的人ODFcDNA序列与文献报道的核苷酸序列一致 ,Western印迹证实稳定转染人ODF的COS 7细胞系中有ODF的表达。结论 构建了人破骨细胞分化因子 (ODF)的真核表达载体 ,并在COS 7细胞中获得稳定表达。

Objective To clone the encoding gene of human osteoclast differentiation factor (ODF),to construct recombinant human ODF mammalian cell expression vector,and to express the gene in COS-7 cells.Methods Using the isolated total RNA from osteosarcoma cell line MG63 as a template,the cDNA encoding hODF was amplified by reverse transcription-polymerase chain reaction method with upstream primer containing Kozak sequence and KpnⅠsite,and down stream primer containing XhoⅠsite.The PCR product was digested with KpnⅠand XhoⅠ,and cloned into eukaryotic expression vector pcDNA 3.1(+) and then sequenced.Finally,the constructed recombinant plasmid pcDNA3.1(+)-ODF was transformed into COS-7 cell line and ODF expression in cell line COS-7 was determined by Western blotting. Results The sequence of ODF cDNA obtained in this experiment was the same as that of reported previously.The recombinant plasmid pcDNA3.1(+)-ODF was constructed successfully and transformed into COS-7 cell line.Secretive recombinant human ODF was detectable in the supernatant of transfected COS-7 cells by Western blotting.Conclusion ODF cDNA is obtained and its expression in cell line COS-7 is determined.These results enable to further study on the function of hODF.