大鼠表皮生长因子、睫状神经营养因子真核表达载体的构建及其体外表达的鉴定

背景：星形胶质细胞被激活后表现出神经干细胞的特性,细胞表面的神经营养因子(表皮生长因子、睫状神经营养因子)受体超表达,通过改善复杂的内环境,有利于定向诱导神经干细胞向神经元的分化。目的：构建大鼠pSecTag2/Hygro B-EGF、pSecTag2/Hygro B-CNTF真核表达质粒,检测其在cos-7细胞中的共表达。方法：采用反转录-聚合酶链反应技术从大鼠颌下腺、坐骨神经组织总RNA中扩增出表皮生长因子、睫状神经营养因子基因功能区,将上述基因片段分别连接到真核表达载体pSecTag2/Hygro B,聚合酶链反应初步筛选、双酶切鉴定后送测序。将构建成功的两种重组载体单独及共转染cos-7细胞,Western blot法鉴定重组表皮生长因子、睫状神经营养因子蛋白的瞬时表达。结果与结论：反转录-聚合酶链反应结果证实成功获得大鼠表皮生长因子、睫状神经营养因子cDNA。DNA序列分析证实2种真核表达载体中的表皮生长因子、睫状神经营养因子序列与GenBank中目的序列一致。脂质体介导转染cos-7细胞48 h后,Western blot鉴定重组表皮生长因子、睫状神经营养因子蛋白在cos-7细胞中的表达,分别在Mr6 000,22 000处出现阳性条带。提示大鼠表皮生长因子、睫状神经营养因子基因的真核表达载体pSecTag2/Hygro B-EGF、pSecTag2/Hygro B-CNTF构建成功,共转染cos-7细胞后能够共表达重组表皮生长因子、睫状神经营养因子蛋白。

Construction of eukaryotic expression vector of rat epidermal growth factor and ciliary neurotrophic factor and their expression in cos-7 cells

BACKGROUND:After activation, astrocytes exhibit the characteristics of neural stem cells and overexpress the receptor of epidermal growth factor (EGF), ciliary neurotrophic factor (CNTF), which improves complex internal environment and therefore benefits for neuronal differentiation of neural stem cells.OBJECTIVE:To construct the eukaryotic expression vectors of pSecTag2/Hygro B-EGF and pSecTag2/Hygro B-CNTF, and detect EGF and CNTF expression in cos-7 cells so as to provide experimental evidence for gene therapy on spinal cord injury.METHODS:The cDNA fragments of EGF and CNTF genes were amplified from total RNAs respectively. The amplified fragments were respectively inserted into eukaryotic expression vector pSecTag2/Hygro B to construct the recombined plasmid that encoded EGF and CNTF cDNA. The plasmids carrying EGF and CNTF genes were transfected alone respectively or cotransfected into cos-7 cells by liposome method. Then the protein expressions were detected by western blot method.RESULTS AND CONCLUSION:RT-PCR results confirmed that EGF and CNTF cDNAs were successfully cloned. DNA sequence analysis confirmed that EGF and CNTF cDNAs in the constructed vectors were consistent with target sequences in the GenBank. Then two recombinant plasmids were cotransfected into cos-7 cells by liposome reagent. At 48 hours after transfection, EGF and CNTF protein expressions in cos-7 cells with the molecular weight of Mr6 000, 22 000 were identified by western blot analysis. These findings suggest that the eukaryotic expression vectors of pSecTag2/Hygro B-EGF and pSecTag2/Hygro B-CNTF were successfully constructed and they co-express EGF and CNTF after transfected into cos-7 cells.