生长分化因子5基因真核表达质粒pcDNA3.1(+)/hGDF5的构建、鉴定及其活性检测

Construction identification and activity assay of eukaryotic expression plasmid pcDNA3.1(+)/hGDF5 containing growth/differentiation factor 5 gene

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pc DNA3.1(+)/h GDF5,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p CA350-h GDF5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pc DNA3.1(+)质粒连接构建真核表达质粒pc DNA3.1(+)/h GDF5。pc DNA3.1(+)/h GDF5经限制性内切酶消化、PCR扩增进行鉴定。将pc DNA3.1(+)/h GDF5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 m RNA及蛋白的表达。结果真核表达质粒pc DNA3.1(+)/h GDF5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pc DNA3.1(+)/h GDF5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pc DNA3.1(+)/h GDF5,为进一步研究GDF5的功能奠定了基础。

Objective To construct and identify eukaryotic expression plasmid pc DNA3. 1（ ＋） / h GDF5 containing growth / differentiation factor 5 gene. The activity of pc DNA3. 1（ ＋） / h GDF5 was detected. Met-hods According to the sequence of GDF5 in Genbank,the primers were designed and synthesized. The product was cloned with pc DNA3. 1（ ＋） vector after amplification with polymerase chain reaction（ PCR）. The eukaryotic expression plasmid pc DNA3. 1（ ＋） / h GDF5 was identified by double restrictive edonuclease and PCR. The eukaryotic expression plasmids were transferred into MC615 and 293 cells. The expression of GDP5 m RAN and protein were detected by PCR,Western blotting and immunofluorescence. Results The eukaryotic expression plasmid pc DNA3. 1（ ＋） /h GDF5 was successfully constructed and identified by double restrictive edonuclease digestion and PCR. The expression of GDF5 m RAN and protein could be detected in MC615 and 293 cells by PCR,Western blotting and immunofluorescence. Conclusion The eukaryotic expression plasmid pc DNA3. 1（ ＋） / h GDF5 was successfully established,which lays the foundation for the function research of GDP5.