Objective To construct and identify eukaryotic expression plasmid pcDNA3.1(+)/hGDF5 con-taining growth/differentiation factor 5 gene.The activity of pcDNA3.1 (+)/hGDF5 was detected.Met-hods According to the sequence of GDF5 in Genbank,the primers were designed and synthesized.The product was cloned with pcDNA3.1(+)vector after amplification with polymerase chain reaction (PCR). The eukaryotic expression plasmid pcDNA3.1(+)/hGDF5 was identified by double restrictive edonuclease and PCR.The eukaryotic expression plasmids were transferred into MC615 and 293 cells.The expression of GDP5 mRAN and protein were detected by PCR,Western blotting and immunofluorescence.Results The eukaryotic expression plasmid pcDNA3.1(+)/hGDF5 was successfully constructed and identified by double restrictive edonuclease digestion and PCR.The expression of GDF5 mRAN and protein could be de-tected in MC615 and 293 cells by PCR,Western blotting and immunofluorescence.Conclusion The eu-karyotic expression plasmid pcDNA3.1 (+)/hGDF5 was successfully established, which lays the foundation for the function research of GDP5 .%目的：构建生长分化因子5（growth／differentiation factor 5，GDF5）基因的真核表达质粒 pcDNA3．1（＋）／hGDF5，并对其进行鉴定和活性检测。方法根据 Genbank中人 GDF5序列设计并合成引物，以 pCA350-hGDF5质粒为模板进行聚合酶链式反应（polymerase chain reaction，PCR）扩增获得 GDF5基因，并与 pcDNA3．1（＋）质粒连接构建真核表达质粒 pcDNA3．1（＋）／hGDF5。pcDNA3．1（＋）／hGDF5经限制性内切酶消化、PCR扩增进行鉴定。将 pcDNA3．1（＋）／hGDF5分别转染 MC615细胞及293细胞，利用 PCR、Western blotting及免疫荧光法检测 GDF5 mRNA及蛋白的表达。结果真核表达质粒 pcDNA3．1（＋）／hGDF5经限制性内切酶酶切、PCR扩增表明构建成功，PCR、Western blotting及免疫荧光实验结果显示真核表达质粒 pcDNA3．1（＋）／hGDF5能在MC615细胞及293细胞中稳定表达。结论成功构建了真核表达质粒 pcDNA3．1（＋）／hGDF5，为进一步研究GDF5的功能奠定了基础。
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