杨治,张铭,刘林,彭侃,许鹏

• 1:西安交通大学医学院附属红会医院关节外科

摘要(Abstract)：

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。

关键词(KeyWords)： 生长分化因子5;质粒;活性;鉴定

Abstract:

Keywords:

基金项目(Foundation): 陕西省科技计划项目(2013K12-17-03)

作者(Author): 杨治,张铭,刘林,彭侃,许鹏

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