人真核表达质粒载体pcDNA3.1-hOPG的构建及体外表达

目的 构建表达人骨保护素基因（hOPG）的真核表达质粒pcDNA3．1-hOPG，并检测其在体外的表达，为治疗破骨细胞功能异常引起的骨吸收提供实验基础。方法 从MGC：29565上获得hOPG基因片段并用PCR方法扩增，将其连接于真核表达质粒pcDNA3．1（-），测定序列后，用脂质体包裹转染C2C12细胞，采用免疫组化和骨吸收抑制实验检测OPG的表达及功能。结果 经酶切和测序鉴定证实本实验构建的重组表达质粒正确，该质粒在体外转染C2C12细胞后可表达功能性OPG蛋白。结论成功构建的pcDNA3．1-hOPG重组质粒能在体外表达OPG蛋白。

Construction of eukaryotic expression bicistron plasmid vector pcDNA3.1-hOPG and its expression in vitro

Objective To construct eukaryotic expression bicistron plasmid vector pcDNA3.1-hOPG,and to detect the expression of the plasmid in vitro.Methods hOPG cDNA fragment was extracted from plasmid MGC:29565,amplified by PCR,and inserted into pcDNA3.1(-)vector,then identified by restriction endonuclease digestion and nucleotide sequencing.C2C12 cells were transfected with the plasmid using Lipofectamine 2000.The expression of OPG protein was detected by immunohistochemistry techniques and bone wafer pit assay.Results The constructed recombinant plasmid contained the sequence of hOPG gene.After transfection with the plasmid,active OPG protein could be expressed in C2C12 cells.Conclusion The constructed eukaryotic expression bicistron plasmid vector pcDNA3.1-hOPG can express OPG in vitro,which is the basis for further study on the treatment of bone absorption caused by abnormal activity of osteoclasts.